AN ABSTRACT OF THE THESIS OF
Thomas J. S. Merritt for the degree of Masters of Science in Zoology presented on
December 1. 1994. Title: Regulation of the Development of Sex-Specific Genital
Muscles by the doublesex Gene.
Abstract approved:
Redacted for Privacy
Barbara J. Taylor
To determine the role of doublesex (dsx) in the regulation of the development of
sex-specific musculature, we have examined the development of a set of sexually
dimorphic genital muscles. In both adult males and females ten muscles attach to the
genitalia and terminal segments in sex-specific patterns. Six of these genital muscles
in males and seven in females consistently express B-galactosidase from a
P[79Bactin -lacZJ construct.
XY and XX dsx mutants that develop as intersexes possess both male and female
genitalia. In both XX and XY dsx- , and XX dsx-dominant, intersexes, we fmd the
same subset of male and female genital muscles. Unlike muscle staining in wildtype
flies, staining for B-galactosidase in the intersexes is irregular, suggesting that
expression of the P[79Bactin-lacZ1 construct is a separate phenotype from muscle
presence. In total, we fmd approximately nine genital muscles in the dsx intersexes,
similar to the number of muscles found in either male or female wildtype flies. The
failure of nearly half of the possible male and female genital muscles to form may be
due to the absence of appropriate attachment points on the cuticle or to a limiting
number of muscle precursor cells. From the similar pattern of muscles in the two
different types of dsx mutant intersexes, we conclude that dsx+ function directs the
development of the genital muscles, acting in wildtype flies to repress the
development of muscles of the inappropriate sex.
Lastly, I describe a set of putative myoblasts that are likely candidates for the
precursors of the genital muscles. A similar set of putative myoblasts is found in
male, female and intersexual discs, suggesting that the myoblasts act as a single
primordia for the genital muscles.
Regulation of the Development of Sex-Specific Genital
Muscles by the doublesex Gene
by
Thomas J. S. Merritt
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Master of Science
Completed December 1, 1994
Commencement June 1995
Master of Science thesis of Thomas J. S. Merritt presented on December 1. 1994
APPROVED:
Redacted for Privacy
jor Prof es, rep$FiAting Zoology
Redacted for Privacy
Chair of D
ment of Zatilogy
Redacted for Privacy
Dean of Grad
chool
I understand that my thesis will become part of the permanent collection of Oregon
State University libraries. My signature below authorizes release of my thesis to any
reader upon request.
Redacted for Privacy
C9Ziek S
Thomas J. S. Merritt, Author
ACKNOWLEDGMENTS
First, I'd like to acknowledge, and thank, my advisor, Barbara Taylor, for
funding, guidance and direction. This work, and my education while at OSU, owe a
great deal to her enthusiasm for science and the depth and breadth of her knowledge.
I would like to thank the members of my committee, Carol Rivin, John Morris and
Belinda King, for doing all those things that committees are supposed to do, and
wading through a deep (and long) stream of bad punctuation and misspellings.
I'd also like to thank Laura Knittel for help and discussion in the lab, occasionally
finding my glasses, and listening to my heated reactions to things I heard on the
phone.
Finally, I would like to thank Glen Davis, Nick Geist, Randy Bender, and assorted
other graduate students, staff and faculty for scientific, moral and social support.
And Greg Little for supplying a home away from home that has kept many a lost soul
out of the loony bin. I think I'll have another pint of Sierra before I go.
TABLE OF CONTENTS
page
1. An Introduction to Sex Determination and Muscle Development in
Drosophila melanogaster
1
General Overview of Thesis
1
Description of sexually dimorphic characteristics
2
General model for somatic sexual differentiation
7
Muscle development in Drosophila melanogaster
23
Summary
37
2. The Role of the doublesex Gene in the Determination of the Male and
Female Genital Muscles of Drosophila melanogaster
39
Introduction
40
Materials and Methods
44
Results
48
Discussion
81
3. Identification of Putative Genital Muscle Precursor Cells in Wildtype and
dsx-Dominant Genital Discs
91
Introduction
92
Materials and Methods
95
Results
101
Discussion
104
4. A Summary of Results and Conclusions
109
Bibliography
114
Appendix
122
LIST OF FIGURES
Figure
page
1.1
Summary of the sex-determination gene cascade.
8
2.1
Composite photomicrographs of adult wildtype terminalia.
49
2.2
Schematic drawings of adult wildtype male and female
terminalia.
51
2.3
Composite photomicrographs of adult, dsx- mutant, intersexual
terminalia.
58
2.4
Schematic drawing of adult dsx-mutant terminalia.
62
3.1
Photomicrograph of genital discs from third instar larvae.
97
3.2
Photomicrograph of genital discs from white pre-pupae
99
LIST OF APPENDIX FIGURES
Figure
page
A.1
125
A.2
126
A.3
127
A.4
129
A.5
130
A.6
131
A.7
132
A.8
134
A.9
135
Chapter 1
An Introduction to Sex Determination
and Muscle Development in Drosophila melanogaster
General overview of thesis
Development of sex-specific structures and behaviors is an attractive system in
which to investigate the genetic mechanisms that control adult differentiation. Sex-
specific structures are not needed for individual viability, therefore, mutations that
cause sexual transformation can be studied throughout the life cycle. Further, genetic
and molecular studies have shown that, in Drosophila melanogaster, sexdetermination is a function of a relatively small number of genes, making Drosophila
a particularly attractive system in which to study the genes involved in establishing a
male or female developmental pathway for sexually dimorphic tissues (recent reviews:
Slee and Bownes, 1990; Steinmann-Zwicky et al., 1990; Burtis and Wolfner, 1992;
Burtis, 1993).
I have investigated the role of one of the sex-determining genes, doublesex (dsx),
in controlling the development of a set of sex-specific muscles in Drosophila
melanogaster. The muscles I have studied surround and/or attach to the internal and
external genitalia in a stereotypic, sex-specific pattern. During the metamorphosis
from the larva to the adult, larval muscles histolyse and adult skeletal muscles arise
de novo from muscle precursor cells set aside in the embryo. The final morphology
and location of the genital muscles must then depend on the activity of multiple genes
that, as a group, regulate both muscle differentiation and sexual differentiation.
2
In the first half of this chapter I describe sex-specific differences in Drosophila
and give an overview of the genetic control of the development of these
characteristics. In the second half of this chapter I review muscle development in
Drosophila. In the second chapter, I describe, in detail, the genital muscles in
wildtype and dsx mutant flies and discuss the role of dsx in development of these
muscles. In the third chapter, I briefly describe the location of putative myoblasts in
the genital disc, a possible source of the genital muscles. In the fourth, and
concluding, chapter, I speculate on future directions for this research.
Description of sexually dimorphic characteristics
The two sexes of Drosophila differ in many external and internal structures
(Ferris, 1950; Miller, 1950), and in the production of sex-specific molecules, such as
pheromones and yolk proteins (see, for example, Burtis and Wolfner, 1992; recent
reviews: Slew and Bownes, 1990; Steinmann-Zwicky et al., 1990; Burtis and Wolfner,
1992; Burtis, 1993). Furthermore, each sex displays specific courtship and mating
behaviors (see, for example, Taylor et al., 1994). The adult sex-specific differences
are first visible in larvas as growth differences in imaginal tissues. Final elaboration
and differentiation of these sex-specific imaginal tissues occurs during metamorphosis.
Sex-specific differences in the germ line and its control, while of great import to the
individual and the species, will not be addressed here (but, see for recent review,
Steinmann-Zwicky, 1992; Gorman and Baker, 1994).
3
The body of an adult fly is divided into three distinct regions: head, thorax and
abdomen. Most sex-specific differences are associated with the abdomen, but sets of
sex-specific chemo- and mechano-sensory bristles are present on the head and
forelegs. Drosophila have six legs, each leg has ten segments: the coxa, trochanter,
femur, tibia and five tarsal segments. On the first tarsal segment of the prothoracic
(most anterior) leg, males have a row of large, right-angled, blunt, heavily pigmented
bristles, known as the sex comb (Ferris, 1950; Tokunaga, 1962). In females the
same tarsal segment has two rows of bristles, identical in size, angle and pigmentation
to bristles on the other legs. Additionally, along the entire prothoracic leg the
number and organization of gustatory bristles is also sex-specific; males have a
greater number of bristles than females (Possidente and Murphey, 1989).
Cranially,
and finally, while not as outwardly sexually dimorphic as in some insects, D.
melanogaster antenna do show sex-specific distributions of chemosensilla (Stocker and
Gendre, 1988).
The adult abdomen is segmented; most segments have a dorsal cuticular plate, the
tergite, and a ventral cuticular plate, the sternite. Abdominal sexual dimorphisms
include the number of segments, segment pigmentation, and, most posteriorly, the
genitalia and analia. Males have six large, serially repeated, abdominal segments
(A1 -A6); females have seven (A1 -A7). In males, the tergites of the fifth and sixth
segments are uniformly pigmented, completely dark. In females, all tergites are only
pigmented across their posterior region, producing a banded pattern. The genital and
anal structures are located at the caudal end of the abdomen.
4
These anal and genital structures are the most dramatic of the sexual differences.
Two laterally paired, equal-sized, cuticular plates cover the anus of male flies, in
females this pair of anal plates is dorsal-ventral, with a larger dorsal, than ventral,
plate. Centrally, the penis is located within the penis apparatus, ventral to the anal
plates. The external male genitalia includes the genital arch, lateral plates and the
hypandrium, these all surround the anal plates and penis apparauts. In females, a
small dorsal eighth tergite surrounds the anal plates and extends to either side of the
two cuticular structures covering the vulva, the vaginal plates. Both male and female
analia and external genitalia are covered with bristles in specific patterns (Taylor,
1992).
The genitalia and analia develop from a single imaginal disc, the genital disc,
attached along the ventral midline in the posterior of both male and female larvae.
Gynandromorph and somatic recombination studies (Nothiger et al., 1977; Schupbach
et al., 1978; Epper and Nothiger, 1982) as well as metamorphosis of disc fragments
(Epper, 1981; Epper, 1983a; Epper and Bryant, 1983), have shown that this imaginal
disc contains three separate primordia, a male primordium, a female primordium and
an anal primordium, in third instar larvae of either sex. Sex specific cell growth, and
death, during metamorphosis results in the final sex-specific adult phenotypes of the
genitalia and analia (Epper, 1983b; Taylor, 1989a).
The male primordium is in the anterior and lateral region of the disc; in males this
region differentiates into the set of internal and external structures that make up the
male reproductive system (Epper and Nothiger, 1982; Taylor, 1989a). In females,
5
the cells of this region divide very slowly during larval life and eventually die during
metamorphosis (Epper and Nothiger, 1982; Epper, 1983b; Taylor, 1989a). The
female primordium occupies the ventral region of the disc, in females it develops into
the external and internal genitalia; in males this region degenerates (Epper and
Nothiger, 1982; Epper, 1983b; Taylor, 1989a). The single anal primordia is located
the dorsal posterior-most region of the disc. In females the anal primordia gives rise
to the dorsal and a ventral anal plates. In males the anal primordia, or a subset of
this region, gives rise to a pair of lateral anal plates (Epper and Bryant, 1982; Taylor,
1989a).
Internal sexual dimorphisms have been described in the musculature, nervous
system and fat body. Given the pronounced differences in the external genitalia it is
not surprising that there are different genital muscles in males and females. These
muscles have previously been incompletely cataloged in descriptions of adult
abdominal cuticle and musculature (Ferris, 1951; Miller, 1951). In the next chapter I
give a more complete description of these genital muscles. To date, only one other
sex-specific muscle has been reported, the Muscle of Lawrence (MOL), found in the
fifth abdominal segment of adult males, but not adult females (Lawrence and
Johnston, 1984). This bilaterally paired muscle lies lateral to the dorsal midline,
spanning nearly the full extent of the fifth abdominal segment.
As required by their respective reproductive tasks, males and females perform
distinctive behaviors, (for review, Speith, 1974). Males have a behavioral repertoire
associated with precopulatory and copulatory events. Females, on the other hand,
6
accept or reject courtship, engage in copulation and lay eggs on suitable substrates.
While control of these behaviors has been mapped to the CNS using gynandromorphs
(Hall, 1977; Hall, 1979; Tompkins and Hall, 1983), only a few specific
neuroanatomical sexual dimorphisms have been described. Sex-specific nerve
terminals in the central nervous system are associated with the sex-specific sensory
bristles described above (Possidente and Murphey, 1989; Taylor, 1989b).
Additionally, a higher order olfactory processing center, the mushroom body, has a
different number of fibers in males and females (Technau, 1984). However, no role
in courtship has been demonstrated for this brain region; males with severe
mushroom-body defects still court in an apparently normal fashion (Heisenberg, 1980;
deBelle and Heisenberg, 1994). Instead these regions appear to be involved in
olfactory learning (deBelle and Heisenberg, 1994), a reason for the sex-specific
morphology has not been found. Another sex-specific difference has been described
in a dozen abdominal neuroblasts. Male neuroblasts have more divisions during
larval and early pupal stages than female neuroblasts, resulting in upwards of 20
additional neurons per neuroblast in an adult male than an adult female (Taylor and
Truman, 1992). But, again, no specific function for these neurons has been
described.
Males and females also differ in some of the biochemical products they produce.
Among the compounds specifically made by males are those made by the male
accessory gland, an internal genital organ derived from the male primordia of the
genital disc, and transferred to females during mating; several of the proteins act to
7
alter subsequent behaviors by inseminated females (see, for example, Kubli, 1992).
Females produce long-chain hydrocarbons that act as mate attractants, these molecules
are produces in much smaller quantities by males (for review of chemical
communication in Drosophila see la Ron, 1984). In Drosophila, some of the proteins
that make up the egg yolk are produced in the female fat bodies and transported to the
gonads, and developing eggs, through the hemolymph. Male fat bodies do not
normally produce yolk proteins (Ota et al.,1981; Coshigano and Wensink, 1993;
reviewed by Bownes, 1994; Burtis and Wolfner, 1992). Further, a set of malespecific RNA's, the male-specific transcripts (mst's) are produced by the male
accessory gland (Di Benedetto, 1987). The glucose dehydrogenase gene (Gld) is also
expressed in a sex-specific pattern during development of both male and female
genitalia (Feng et.al., 1991).
General model for somatic sexual differentiation
The somatic differentiation of Drosophila melanogaster into either a male or
female morphology depends on the activity of a gene cascade transmitting information
on the chromosome complement to downstream genes responsible for sex-
differentiation (Figure 1; recent reviews: Baker, 1989; Slee and Bownes, 1990;
Steinmann-Zwicky et al., 1990; Belote, 1992; Burtis and Wolfner, 1992; Cline,
1993). Female Drosophila are homogametic, XX, males are heterogametic, XY.
Unlike the situation in mammals, it is the ratio of X chromosomes to autosomes (X:A
8
ratio) that determines sex, not the presence or absence of a sex-determining
chromosome (Bridges, 1921; Cline, 1983; 1988; reviewed by Hodgkin, 1992; Cline
1993; Gormanand Baker, 1994). Females, with two X chromosomes and a diploid
set of autosomes, have a X:A ratio of 1; males, with only a single X have a ratio of
.5.
female:
XX:2A flies
male:
XY:2A flies
sis-a, sis-b, dpn, da, emc, run
Sxl active
tra active
tra-2 active
dsx active
DSX' produced
Female Differentiation
Sxl inactive
tra active
tra-2 active
dsx active
DSXM produced
Male Differentiation
Figure 1 Summary of the sex-determination gene cascade.
In the somatic tissues, decisions on sexual fate are predominantly cell-autonomous.
This autonomy is apparent in mosaic flies in which the soma is a mix of both
chromosomally male (XO) and chromosomally female (XX) cells (eg Nothiger et al.,
1977). Individual cells express the sexual fate, male or female, appropriate for their
9
chromosomal state, not that of their neighbors. The fifth segment male-specific
muscle, the MOL, is an important exception to this cell-autonomous control of sexdetermination (Lawrence and Johnston, 1986), as will be discussed later.
Control of sex-determination in Drosophila can be likened to a series of on/off
switches: on, female differentiation; off, male differentiation (reviews: Baker, 1989;
Slee and Bownes, 1990; Steinmann-Zwicky et al., 1990; Belote, 1992; Burtis and
Wolfner, 1992; Cline, 1993). Each switch is set by the presence, or absence, of a
functional product from the previous gene in the cascade. The product of one gene
influences the pattern of splicing of the next gene in the cascade, so that a common
pre-mRNA is differentially spliced to yield sex-specific transcripts. These sexspecific transcripts are then translated into functional proteins, in one, or both, of the
sexes, which then influence the splicing of the next gene. In most cases the outcome
of differential splicing produces functional protein in only one sex, but at the output
level of the cascade both spliced products yield active protein products.
The gene cascade consists of five known genes; Sex-lethal (Sxl), transformer (tra),
transformer-2 (tra-2), doublesex (dsx) and intersex (ix). In female Drosophila, an
active, female-specific, Sxl gene product is produced (Figure 1). This protein
promotes female-specific splicing of the tra primary transcript. The female-specific
transcript is then translated into an active, female-specific, TRA protein. The tra-2
protein, TRA, is found in both sexes. In females, the TRA and TRA-2 proteins act
together to control splicing of the dsx primary transcript. The female-specific dsx
protein, DSr, along with the product of the ix gene, represses male differentiation
10
and promotes female differentiation. In males, where no active Sxl protein is
produced, the tra pre-mRNA is spliced in a default pattern that does not encode a
protein. In the absence of TRA protein, dsx pre-mRNA is spliced into the malespecific mRNA. In contrast to the male-specific Sxl and tra transcripts, male-specific
dsx mRNA encodes a functional protein, DSXM, that is required for normal male
development.
The Sex lethal gene
At the top of the genetic cascade, Sxl, regulates dosage compensation, as well as
somatic and germline sex-determination. To maintain equal levels of X chromosome
gene products in both sexes, transcription from the single X in males is boosted to
twice the level of a either X in females (reviewed by Hodgkin, 1992; Gorman and
Baker, 1994). The SXL protein prevents this hypertranscription in females. Null
mutations in Sxl are generally female lethal due to a failure to prevent
hypertranscription of the X-chromosomes (Marshal and Whittle, 1978; Cline 1980;
1983). When the sex-determining function of Sxl is disrupted, without destroying its
dosage compensation function, XX flies develop as somatic males (Cline, 1984). XY
Sxt flies develop as normal males, in fact, Sxl activity is deleterious to males,
preventing the hypertranscription of their single X chromosome (Marshal and Whittle,
1978; Cline 1980; 1983).
Sxl activity is initially directed by transcriptional activation early in development
(Salz et al., 1989; Keyes et al., 1992). The Sxl gene has two promoters, an early
11
promotor, PB and a late promotor, PL. Early production of Sxl protein appears to be
a function of use of the stage- and sex-specific promotor, Pa. Transcripts from PB are
spliced in a female-specific pattern, leading to the functional protein. Transcription is
initiated from PE only in females and only during a brief interval during the syncytial
blastoderm stage. The mechanism restricting the use of PE is as yet incompletely
understood, but one model for Sxl activation, involving both maternal and zygotic
factors, seems to best fit the currently available data (Cline, 1988; 1993; Slee and
Bownes, 1990; Steinmann-Zwicky et al., 1990; Belote, 1992). In this model the ratio
of certain X-linked genes, numerator genes, to some autosomal genes, denominator
genes, determines the activity of the Sxl gene. This ratio is transmitted to the Sxl
gene by a set of maternally and zygotically transcribed gene products.
Mutations in
genes acting as either numerators or denominators would result in errors in dosage
compensation, which, in turn, would result in zygotes of only one sex. Two genes,
sisterless-a and sisterless-b (sis-a, sis-b), have been identified as numerator genes
(Cline, 1988). Whereas the gene deadpan (dpn) appears to be the only denominator
gene (Younger-Shepherd et al., 1992).
Sex-lethal activity also requires the maternal function of at least two other genes,
daughterless (da; Cline, 1980; 1983) and extramacrochaete (emc; Younger-Shepherd
et al., 1992). While da and emc are required for proper sex-determination and
dosage compensation, they are not required in a dosage specific manner. They are
not, therefore, numerator or denominator genes, but, rather, function through some
other role, possibly in transmitting the numerator:denominator ratio to Sxl.
12
Zygotically transcribed runt gene product is also required in a non-dosage dependent
manner for proper Sxl activity (Torres and Sanchez, 1992).
Molecularly, control of Sxl activity is thought to involve protein-protein
interactions which titrate certain DNA binding proteins. sis-b, dpn, da and emc all
code for proteins with sequences consistent with a helix- loop -helix (HLH) secondary
structure (Younger-Shepard et al., 1992). This structure is characteristic of proteins
that bind DNA as hetero- or homo-dimers (Murre et al., 1989). The activity of these
proteins may be a function of their binding to the Sxl gene and/or each other.
Following the syncytial blastoderm stage, in both sexes transcription shifts from
the from the PE promoter to the PL promotor. This later transcription only leads to
production of functional protein in females. In females a Sxl transcript is present that
does not contain the third exon and does contain a long ORF (Bell, 1988). This
transcript codes for a protein similar to that produced from the PE transcription
product. In males all Sxl primary transcripts, all transcribed from the PL promotor,
contain a third exon containing multiple stop codons. These transcripts do not contain
any long ORF and no Sxl protein is produced (Bell, 1988).
An autoregulatory mechanism ensures that functional Sxl protein continues to be
made in females; once active Sxl protein has been produced, from PE during normal
female development, it is both necessary and sufficient to regulate the continued
production of active Sxl protein, irrespective of future chromosome complement.
SXL binds to its own transcript, promoting female-specific splicing (Bell et al., 1991;
Sakomoto et al., 1992). This binding is dependent on multiple uridine rich sequences
13
found around the male-specific exon. Deletion of these sequences eliminates female
specific splicing. Female-specific splicing was restored with addition of new poly-U
sequences to the deleted constructs. Similar poly-U sequences are important in Sxl
regulated sex-specific splicing of the transfonner primary mRNA.
Sex-lethal regulation of the transformer gene
transformer is the next gene in the sex-determination regulatory cascade (Baker
and Ridge, 1980, Nagoshi et al., 1988). Active tra gene product is required for
proper female sexual differentiation; XX tra flies develop as phenotypic males
(Sturtevant, 1945). Active TRA is not required in males; XY tra homozygotes
develop as normal males (Baker and Ridge, 1980).
Production of TRA protein is dependent on sex-specific splicing of a non-sexspecific tra pre-mRNA into sex-specific transcripts (Boggs et al., 1987; Mckeown et
al., 1988). This splicing is controlled by SXL binding to specific sequences within
the tra pre-mRNA in a manner similar to that in which SXL controls splicing of its
own transcript (Sosnowski et al., 1989; Inoue et al., 1990; Valcarcel et al., 1993).
Thus, two tra mRNA transcripts are found in adult Drosophila. These transcripts
vary in the first exon splice acceptor site used; use of the upstream site produces a
mRNA found in both sexes. Whereas use of the downstream site produces a mRNA
only found in females (Boggs et al., 1987). Only this female specific transcript
contains a long ORF and produces functional tra protein (McKeown et al. 1988).
14
Potentially, Sxl could control splicing of tra pre-mRNA by either promoting the
use of the 3', female-specific, splice acceptor site or blocking the use of the 3', nonspecific, site. Tests of the site of SXL action showed that deletion of the non-sexspecific splice site leads to Sxl- independent use of the female-specific splice site in
vivo (Sosnowski et al., 1989). In vitro, the Sxl protein binds specifically to poly-U
sequences around the non-specific splice site (Inoue et al., 1990, Valcarcel et al.,
1993), similar to the poly-U sequences near exon 3 of the Sxl transcript that are
involved in Sxl self-regulation (Sakamoto et al., 1992). Binding of s)a, to these
sequences prevents formation of the spliceosome at the non-specific splice acceptor
site and the female-specific site is used by default (Valcarcel et al., 1993). This
strongly suggests that in vivo Sxl mediated control of tra splicing is a function of the
SXL protein binding directly to the splice acceptor site within the non-sex-specific tra
exon blocking its use and preventing inclusion of this exon in the tra final transcript.
The transformer -2 gene
tra-2 loss-of-function mutants have a phenotype very similar to tra loss-of-function
mutants (review, Baker and Belote, 1983). The transformer-2 protein acts, along
with the product of the transformer gene, to promote female-specific splicing of the
doublesex primary transcript (Baker and Ridge, 1980; Belote and Baker, 1982;
Nagoshi et al., 1988). Like the TRA protein, the TRA-2 protein is required
continuously in the female soma for normal sexual development, but is not required
for normal male somatic development (Belote and Baker, 1982). Unlike the TRA
15
protein, the TRA-2 protein is required for proper spermatogenesis in the male germ
line (Belote and Baker, 1982). A functionally similar TRA-2 protein is found in the
soma of both sexes, but influences
dsx
pre-mRNA splicing only in the presence of
functional TRA protein.
Molecular evidence reveals a possible mechanism of action. The TRA-2 protein
shows noted sequence homology to a family of RNA binding proteins that includes
hnRNPs and snRNPs, suggesting that tra-2 may act by binding directly to the dsx
transcript (Goralski et al., 1989).
The doublesex gene
Both genetic (Baker and Ridge, 1980) and molecular (Nagoshi et al.,1988) studies
place the doublesex gene (Hildreth 1965) downstream of Sxl,
and
tra-2
are all required for proper
dsx
tra
and
tra-2.
Sxl, tra
activity in females; Sxl acts trough tra,
which along with tra-2 acts directly on dsx.
dsx is
the only sex-determining gene
which is required for normal somatic sexual development of both sexes; both XX or
XY dsx- homozygotes develop as intersexes, expressing a combination of male and
female sex-specific characteristics (Hildreth, 1965; Baker and Ridge, 1980).
The general theme of control through sex-specific splicing is seen in the control of
dsx.
Late in larval development male- and female-specific transcripts first appear
through the alternative splicing and polyadenylation of a common pre-mRNA which
produces transcripts with identical 5' ends, but different 3' ends (Baker and Wolfner,
1988; Burtis and Baker, 1989). Exons 1,2,3 are found in transcripts from both sexes.
16
Exon 4 is found only in female-specific transcripts, while exons 5 and 6 are found
only in male-specific transcripts. These transcripts are translated into sex-specific
proteins with identical carboxy-, but different amino-, terminus, DSXM
and DSr.
Production of the female-specific transcript is the regulated step in control of dsx
activity which is controlled by the activity of the tra and tra-2 genes. In the absence
of either tra, or tra-2, as in males or Sxt, ta' or tra -2' females, the dsx pre-mRNA is
spliced in the male pattern by default. Production of the female-specific transcripts
could be regulated through control at either a splicing event or at the choice of
polyadenylation sites. Multiple investigations of both the splice site and the
polyadenylation site point towards a regulated splicing event. There are four lines of
evidence supporting the regulation of female-acceptor site by TRA and TRA-2
interaction. The female-specific fourth exon splice acceptor site shows poor
homology to the Drosophila consensus splice acceptor site sequence, while the male
exon slice acceptor site shows a good consensus, supporting the possibility that
control could be via discrimination between the two sites (Burtis and Baker, 1989).
Interestingly, a number of dominant mutations have been isolated that effect normal
female sexual development, but do not effect normal male sexual development (Feng
and Gowen, 1957; Baker and Ridge, 1980; NOthiger et al., 1980). XX flies with a
dominant dsx allele over a dsx deficiency (dsx'"Vdsx) develop as somatic males; XY
dsxD'idsx- flies develop as normal males. The aberrations causing these dominant
dsx alleles are all located around the female-specific splice site and distant from the
polyadenylation signal sequence (Baker and Wolfner, 1988; Burtis and Baker, 1989;
17
Nagoshi and Baker, 1990), further implicating splicing and not polyadenylation as the
point of control. Finally, the female-specific fourth exon contains six copies of a 13
nucleotide repeat (Nagoshi and Baker, 1990). All of the dominant dsx alleles delete
or displace the region containing these six repeats (Nagoshi and Baker 1990). In vitro
binding experiments have demonstrated that these sequences are the cis-acting control
elements for sex-specific splicing (Inoue et al., 1992). In cultured Drosophila cells a
dsx "minigene", a construct containing portions of the third, fourth and fifth
exons, is
spliced in the female specific pattern in the presence of the TRA and TRA-2 proteins.
Minigenes lacking a subset of the lint repeats were spliced in the male pattern,
replacement of the repeats rescued female-specific splicing. Further, bacterially
generated TRA and TRA-2 proteins were directly demonstrated to bind to the 13nt
sequences (Inoue et al., 1992; Tian and Maniatis, 1992). Neither protein was found
to bind to mutant 13 nt sequences. Similar results have been reported by other
investigators showing, in vitro, that the 13nt repeats within the female-specific exon
are both necessary and sufficient for female-specific splicing (Ryner and Baker, 1991;
Tian and Maniatis, 1992). Further, and most importantly, deletion of the female
polyadenylation site does not prevent female-specific splicing of the dsx transcript in
vitro (Ryner and Baker, 1991). In total, this strongly suggests that in Drosophila
regulation of female-specific splicing of the dsx transcript by tra and tra -2 involves
control of splicing, not polyadenylation.
Based on the early genetic evidence, and largely substantiated by the later
molecular evidence, a model for dsx function was proposed in which sex-specific dsx
18
proteins acted antagonistically as repressors (Baker and Ridge, 1980; Belote and
Baker, 1982). In this model each DSX protein repressed the development of
characteristics specific to the other sex. If neither & product was present, neither
sexual pathway was repressed, and characteristics of both sexes were expressed, an
intersexual phenotype. If both products were present, as in XX; dsx 'Vdsx+ flies, the
proteins were proposed to canceled each other out, again producing intersexes. This
model holds true for many of the sex-specific characteristics that have been described.
As a prelude and introduction to the possible role of dsx in control of the genital
muscles (to be discussed in the next chapter), I will review and examine the role of
dsx in development of other sex-specific traits.
dsx control of sex-specific phenotypes
Most sex-specific phenotypes that have been studied depend on proper doublesex
activity for their normal development. Production of either DSX' or DSX'
determines which sex-specific phenotype will be repressed and which will develop.
For example, XX flies with mutations in Sxl, tra or tra-2 produce DSX' (Nagoshi et
al., 1988) and show a male pattern of tergite melanization, male sex combs and male
genitalia (Baker and Ridge, 1980). Further, these flies show a male pattern of
gustatory leg bristles and a male pattern (contralateral and ipsilateral projection) of
afferent projections from these bristles (Possidente and Murphey, 1989). XX flies
expressing DSX' also show some male biochemical phenotypes. They express the
19
male-specific pattern of glucose dehydrogenase gene expression (Feng et al., 1991)
and produce male-specific transcripts (msts) in their accessory glands (Chapman and
Wolfner 1988).
Early studies of the effect of dsx mutations on sex-specific phenotypes (i.e. Baker
and Ridge, 1980) suggested that dsx had only a negative regulatory role in
development. Its role was construed as only being required for repression of sexual
characteristics of the opposite sex, but for activation of the appropriate sexual
phenotypes. For example, the sex-combs develop a similar intersexual phenotype in
intersexes resulting from recessive mutations or dominant dsx mutations. The
phenotype of XX dsx', + suggests that lack of either dsx protein appears to have
the same effect as presence of both dsx proteins. This is consistent with the proteins
acting as repressors that can block each others activity, but would not be expected if
one or both of the DSX proteins was required for normal development.
Recently, however, phenotypes have been described that do not fit with this simple
repression model of activity and suggest a further, positive, regulatory role for the
sex-specific DSX proteins. In female fat body cells, yolk proteins are produced and
shipped to the ovaries for egg production via the hemolymph. These proteins are
coded for by three genes, ypl, yp2 and yp3. Two of these genes, ypl and yp2, are
under the control of a single promotor (Barnett et al., 1980; Postlethwait and Jowett,
1980). In wild type males the fat bodies produce no yolk proteins. As would be
expected from a phenotype under dsx control, ectopic expression of DSXM in XX flies
prevents Yp production (Ota et al., 1981). Further, DSXM has been shown, in vitro,
20
to bind directly to a region of the common promotor of ypl and yp2, the fat body
enhancer, FBE. Surprisingly, DSXF also binds to the FBE in vitro. In fact, both
DSXF and DSXM bind to the same three sites within the FBE (Burtis et al., 1991). It
has been recently demonstrated that DSXF is required for wild type levels of protein
production in XX flies (Coshigano and Wensink, 1993). This suggest that control in
vivo is a product of either DSXM binding to the FBE and inhibiting transcription or
DSXF binding to the FBE and promoting transcription. How binding of these two
proteins to the same region of the gene results in opposite effects is not known, but
presumably results from different actions due to the differential carboxy termini of the
two DSX proteins.
dsx also appears to have a positive role in control of male-specific cell division in
one region of the developing CNS. As mentioned, a set of neuroblasts in the terminal
ganglia show a longer period of division in male than in females (Taylor and Truman,
1992). When both DSXM and DSXF are present, individual neuroblasts adopt either
the male or female pathway; they are apparently able to chose either the male or
female sexual pathway. However, when neither protein is present, neuroblasts do not
divide at all, in either sex. DSXM is apparently necessary for any division of neurons
in the male state. Control of the pattern of division, then, is not a function of
repression by DSXF, but of activation by DSXM.
Another example of a possible positive regulatory role for DSXM has recently been
reported. Ectopic expression of DSXM, using hsp70 promotor-dsx cDNA fusion
products, produced a number of unexpected phenotypes (Jursnich and Burtis, 1993).
21
Of most interest was a transformation of leg bristles, in legs which do not normally
have sex-differences, to a sex-comb-like morphology. The presence of DSXM
apparently activated expression of a sex-comb-like morphology in the legs. It was
proposed that sex-comb development is controlled by a gene, or set of genes, under
the control of DSXM. In a situation similar to that of the Yp gene, but with the roles
reversed, DSX1' is proposed to repress gene activity, while DSXM acts to increase it.
Obviously other genes must be involved to regulate proper leg and leg segment
positioning.
Still other cases indicate that dsx may not be involved in sex-determination of all
tissues; two examples of sex-specific characteristics that are unaffected by dsx
mutations have been described. Female flies with loss-of-function mutations of tra
and tra-2, or with mutations effecting the sex-determining functions of Sxl, perform
male courtship behaviors, however, no such transformation is seen in females with
loss-of-function alleles of the dsx or ix genes (Mc Robert and Tompkins, 1985; Taylor
et al., 1994). While XX tra- or tra-2- pseudomales show male courtship, no
mutations in the dsx gene induced XX flies to express any measurable male courtship
phenotypes (Taylor et al., 1994). Similarly, while some dsx mutations do reduce
courtship by XY flies, no dsx alleles ever completely eliminated courtship (Taylor et
al., 1994).
The second dsx-independent phenotype is especially relevant to this study of sex-
specific muscle development. Formation of the fifth segment male muscle, the MOL,
is also independent of dsx or ix activity (Taylor 1992). Transformation of XX flies
22
due to mutations in Sxl, tra or tra -2 includes expression of a MOL; transformation
due to mutations in dsx does not. Likewise, and as with male courtship behavior, XY
flies expressed a MOL regardless of mutations in dsx. If dsx is not involved, how,
then, is sex-specific differentiation of these characters regulated?
Two solutions to resolve this discrepancy seem possible. Either the regulatory
gene cascade branches out, above the level of dsx and ix, to include other genes, or
tra and tra-2 act directly to control expression in a manner similar to the activity of
dsx on other traits (Taylor, 1992; Taylor et al., 1994). As yet, it is not possible to
rule out either of these two possibilities. However, given the specific nature of the
interaction between the TRA and TRA-2 proteins and the repeats in the dsx transcript,
it seems less likely that TRA and TRA-2 would act directly on a number of different
terminal differentiation genes.
One gene, which is known to control MOL development and to cause courtship
defects, has been advanced as a possible candidate for a branch of the sex-
determination gene cascade. Mutations of the fruitless (fru) locus have multiple
effects on male flies (Hall, 1978; For review Hall, 1994). Along with unusual
courtship behaviors fru/fru homozygous males lack, or partially lack, the MOL
(Gailey et al., 1991). What exact role the gene(s) associated with this inversion have
in patterning of the MOL has yet to be determined.
What is the role of dsx in determination of the genital muscles? dsx is not
involved in determination of the MOL, the best studied sex-specific muscle leading to
the question: Are the genital muscles regulated by dsx? We will address this question
23
in the next chapter. Whatever the role of the dsx gene, the development of the genital
muscles is a function of muscle-specific regulation. Before examining the genital
muscles in particular I will review muscle development in Drosophila in general.
Muscle development in Drosophila melanogaster
Drosophila are holometabolous insects; a normal life cycle includes an embryonic,
three larval, a pupal and an adult stage. Larval and adult stages have distinct, and
qualitatively different, stereotyped patterns of muscle underlying their epidermis,
reflecting the extremely different modes of locomotion and roles in reproduction of
these two stages. During the intervening pupal stage drastic changes in cuticular,
muscular and nervous systems occur. This change is accomplished through both
rearrangement of larval tissue and creation of adult structures de novo from imaginal
cells.
In general adult muscles are formed de novo from specific pools of cells set aside
in the embryo (Bate, Rushton and Currie, 1991; Bate, 1991). Larval muscles
histolyse during metamorphosis, except for a set of larval muscles which function in
or directly following eclosion (Crosse ly, 1978; Kimura and Truman, 1990). These
retained larval muscles histolyse early in adult life. Additionally, larval muscles may
act as a template for developing adult muscles (Shatoury, 1956; Fernandes et.al.,
1991). During metamorphosis three larval thoracic muscles, the LOMs, split into six
templates with which new myoblasts fuse to form six adult thoracic muscles, the
24
DLMs (Shatoury, 1956; Fernandes et al., 1991). The larval muscles survive the first
wave of histolysis that destroys most larval muscles, split and then fuse with
myoblasts set aside during embryogenesis for adult muscle formation. In at least two
other insect species a similar set of muscles also develop through the fusion of
myoblasts with a template formed by the remnants of larval muscle (Smit and Velzig,
1986; Cifuentes-Diaz, 1989). Interestingly, however, in Drosophila, at least, these
templates do not appear to be required for proper muscle placement; laser ablation of
the templates in developing pupae does not prevent formation of an adult muscle
(Fernandes, personal communication).
Larval muscle functioning as a template for adult muscle formation, though more
common in other insects, is apparently the exception in Drosophila (Crosse ly, 1978,
Niiesch, 1985). While it is more common for adult Drosophila muscle to develop
entirely from cells set aside in the embryo, this case of the LOMs and DLMs
indicates that the possibility of larval templates for the sex-specific genital muscles
can not, a priori, be ruled out. In fact, in Manducca sexta the genital muscles do
develop from a template of larval muscle remnants (Thorn and Truman, 1989). With
the onset of pupation in this species the larval terminal abdominal muscles largely
histolyse and degenerate. The remaining non-contractile "scaffold" of muscle
remnants forms a sex-specific template through cell loss and rearrangement. During
pupation myoblasts fuse with this scaffold to form the adult complement of sex-
specific genital muscles. In general, however, development of the genitalia of this
species is much more a product of rearrangement of larval tissue than in Drosophila;
25
in Manducca only the reproductive tract develops from genital discs (Thom and
Truman, 1989), while the genital discs form the reproductive tracts and the genitalia
in Drosophila (see above). Development of the adult genital muscles around a larval
template seems unlikely in Drosophila since the external and internal genitalia,
attachment points for the genital muscles, form entirely from imaginal tissue.
In Drosophila, the DLMs and the use of larval templates for adult muscles, is the
exception, not the rule. All other known adult muscles form solely from groups of
cells set aside during embryogenesis (Bate, Rushton and Currie, 1991; Currie and
Bate, 1991). How is the development of these muscles regulated? I will first review
what is known about control of larval muscle development and then move on to
control of adult muscle development.
Larval muscle pattern
Three possible mechanisms have been proposed for the regulation of development
of larval muscles: 1) epidermal induction of the developing mesoderm, 2) autonomous
control of pattern formation by the mesoderm, or 3) induction by the nervous system
(Bate, 1990). In the developing embryo, the earliest observed primordial muscles are
known as muscle precursors, fused doublets and triplets of cells. These precursors
already occupy appropriate positions to later form the complete larval muscle pattern
from their first appearance at eight hours after fertilization, which is prior to the
outgrowth of axons from either sensory or motor neurons. Since the final pattern is
observed prior to innervation, nerve activity or contact does not seem to play a role in
26
initial pattern formation. However, muscle precursors are first observed directly over
the CNS and ectoderm, leaving open the possibility that patterning information could
be transmitted from either of these tissues.
Epidermal differentiation precedes muscle differentiation, myoblasts segregate and
fuse over an already differentiated cuticle (Bate, 1990). The cuticle could, therefore,
provide patterning information to developing muscle. It is known that muscle will not
form if the overlying cuticle is removed. Cuticle attachment points are required late
in muscle differentiation, failure to form the proper pattern following cuticle removal
may only show an inability of the muscle to differentiate, not a lack of determining
information.
Hooper (1986) provides evidence implying that epidermis does not have a
determining role. She showed that the homeotic gene Ultrabithorax (Ubx) has
different apparent zones of action in the epidermis and muscle. Ubx transcripts and
segments affected by Ubx mutations (areas showing ectopic expression of anterior
structures) were offset by approximately 1.5 segments between the two tissue types.
If this were a case of strict epidermal induction the same pattern of transformation in
both tissues would be expected. While inductive communication across a gap of 1.5
segments is possible, its seems more likely that muscle development is not a function
of direct induction by the epidermis.
With evidence against induction by either innervating nerves or the epidermis,
mesoderm-autonomous patterning seems the most likely mechanism for patterning of
larval muscle. However, recent work by Broadie and Bate (1993) on Drosophila
27
embryo synaptogenesis, formation of the connection between neuron and muscle cell,
cautions against a strict autonomous versus induced model of pattern formation, but,
instead promotes a more interactive model. They found distinct innervationindependent and innervation-dependent events in synaptogenesis. The muscle leads
the motor neuron to the synaptic cleft (innervation-independent event) and
subsequently the motor neuron directs the development of the receptive field
(innervation independent events). While certain aspects of muscle pattern formation
may be autonomous, formation of a normal neuromuscular junction, at least, requires
interaction between muscle and neuron.
Adult muscle development
Following pupation adult flies emerge with a morphology vastly different from that
of the larvae (Crosse ly, 1978). A distinct head, thorax and abdomen have developed,
as have various appendages and the genitalia. The segments of the thorax contain a
complex, but non-sex-specific pattern of muscles. In the abdomen the muscle pattern
of the first six segments (Al through A6) is also largely non-sex-specific. An
important exception is the afore mentioned MOL ( Lawrence, 1984). Additionally,
the sexes differ in the muscles that surround the genitalia, the focus of this Masters
Thesis.
Metamorphosis takes approximately 96 hours. As pupariation (formation of the
pupa, which is different from the adult) begins the larval cuticle hardens and forms
the pupal case. By 24 hours after puparium formation (APF) nearly all larval muscles
28
have been broken down and removed through histolysis and phagocytosis (Shatoury,
1956). Nerves also regress to a single major trunk in each hemisegment. With the
exception of the previously mentioned DLM's (Shatoury, 1956; Fernades et.al., 1991)
and the retained larval muscles (Crossley, 1978; Kimura and Truman, 1990) all adult
cuticular muscles develop de novo during adult development (Bate et.al., 1991; Currie
and Bate, 1991).
Adult muscles develop from mesodermal cells (Lawrence and Brower, 1982) set
aside in the embryo (Bate et.al., 1991). In the thorax the muscle precursors are the
adepithelial cells found associated with larval imaginal discs (Lawrence and Brower,
1982; Bate et.al., 1991). In abdominal segments Al through A7 the muscle
precursors are not associated with the precursors of the adult epithelium, but occur as
four separate groups of cells; a ventral, a dorsal and two lateral groups (Bate et.al.,
1991). The precursors of the genital muscles have not previously been described, in
the third chapter I will present some preliminary evidence as to the location of these
precursors within the genital disc.
Adult muscle precursor cells express the twist protein (Bate et.al., 1991), a protein
initially expressed in all presumptive mesoderm (Thisse et.al., 1988), until midway in
adult development. Decline in twist expression by these cells coincides with the
fusion into myoblasts and the beginning of expression of muscle specific proteins
(Currie and Bate 1991).
The twist protein has an interesting role in mesodermal development. It has
primarily been of seen as interesting in regards to its role in embryogenesis, where it
29
is vital for gastrulation; twist- flies fail to form any mesoderm and die at the end of
embryogenesis (Beer et.al., 1987). While the function of the twist protein in
embryogenesis has been relatively well studied (eg. Ip et.al., 1992), its function, if
any, in the muscle precursor cells is unknown. The molecular characterization of the
twist protein does suggest a possible mechanism for the action of the twist protein in
the muscle precursor cells. The twist protein shows sequence homology with known
basic helix-loop-helix (HLH) proteins (Murre et.al., 1989) and is localized to the
nucleus (Thisse, 1988), where it acts as a transcriptional activator in presumptive
mesoderm (Thisse et.al., 1988; Ip et.al., 1992). The HLH protein structure may
indicate a possible DNA binding capability. In the muscle precursor cells the twist
protein may act by binding to certain DNA regions and inactivating certain musclespecific genes or activating certain genes responsible for repressing muscle cell
development. At present this is all only interesting speculation.
During germ band retraction (mid-late embryogenesis) twist expression declines
and becomes restricted to small, segmentally repeated, groups of 8-15 cells, the
muscle precursor cells (Bate et.al., 1991). In developing pupae these cells
proliferate, segregate and form the adult musculature (Curie and Bate, 1991).
During metamorphosis, groups of cells, called histoblasts, proliferate and spread
out to form the new adult cuticle. Nerves grow out directly behind the developing
epidermis. At this time the twist expressing muscle precursors also proliferate and
spread out along the developing nerves (Currie and Bate, 1991). The muscle
precursors migrate with the spreading histoblasts and nerve growth zones. By 24
30
hours APF (after puparium formation) the precursors are expressing muscle specific
protein. By 28 hours they have begun to fuse, forming multinucleate cells. By 65
hours APF the final muscle pattern is established, cell fusion is complete and twist
expression is gone (Currie and Bate, 1991).
Broadie and Bate (1991) used ablation experiments to show that, by the second
instar, the twist-expressing cells form primordia for specific muscle subsets.
Hydroxyurea (HU) is a DNA-synthesis inhibitor that blocks the activity of nucleotide
reductase and kills cells as they pass through S-phase of the cell cycle. Adults
developed from second instar larvae fed HU show ablation of groups of muscles.
Ablations were found to show a quantal pattern, muscles were ablated as groups, not
as single fibers. These groups are thought to correlate to the groups of twistexpressing muscle precursor cells.
How is the development and proliferation of these muscle precursor cells regulated
to give rise to the pattern of muscles seen in the adult?
Adult muscle patterning
In the embryo growth of larval muscles follows complete epidermal formation and
precedes outgrowth of nerve axons (Bate, 1991). In contrast, in the pupae, adult
muscle develops concurrently with both the epidermis and nervous system (Bate
et.al., 1991). In light of this important difference, patterning of adult and larval
muscle may well be a function of different mechanisms. The three possible patterning
mechanisms proposed by Bate (1990) for larval muscle; autonomous information,
31
epidermal induction or neural induction, have been investigated in patterning of adult
muscle.
In the larval thorax the muscle primordia are associated with the imaginal discs
(Lawrence, 1982). However, in the larval abdomen the primordia are not associated
with the imaginal epidermis, the histoblast cells, but are closely associated with the
peripheral nerves. The genital muscles may be an exception to this, as will be
discussed in the third chapter. The association of the abdominal muscle precursors
and the nervous system suggests that the nervous system may have a patterning role in
the development of adult abdominal muscle; positioning or providing the information
that positions the muscle precursors. Three facts argue against this possibility. First,
segregation into the groups of adult-specific twist positive cells happens before
outgrowth of the nervous system (Bate et.al., 1991); the initial pattern of twist
positive cells cannot be a product of induction by the peripheral nervous system.
Second, initial segregation of the cells is normal in daughterless (da) homozygotes
although da/da flies do not form sensory neurons (Bate et.al., 1991). In da/da flies
the final pattern of muscle cells is, however, distorted. This is interesting in light of,
and generally consistent with, the Broadie and Bate (1993) paper on embryonic
synaptogenesis; final pattern formation may be a function of an interaction between
the nervous and muscle systems. Third, and most importantly, ablation of the
innervating tissue does not prevent formation of the non-sex-specific muscle pattern
(discussed below).
32
Similar to the earlier investigation of larval muscle patterning (i.e. Hooper, 1986),
homeotic mutations have been used to investigate the role of the cuticle in patterning
of the twist positive muscle precursors (Greig and Akam, 1993). It was found that
the pattern of adult muscle precursors can be altered without necessarily altering the
overlying cuticle, indicating that the muscle pattern does not require induction from
the epidermis (Greig and Akam, 1993). In normal development the homeotic gene
abdominal-A (abd-A) specifies the development of abdominal segments (Karch et al.,
1990; Macias et al., 1990). Ectopic expression of abd-A in thoracic mesoderm of
transformed larvae resulted in expression of an approximation of the abdominal
pattern of twist-expressing cells (adult muscle precursors) in the thorax, without
altering the thoracic cuticle. These results argue against mesodermal dependence on
ectodermal induction for proper development. However, the converse experiment,
ectopic expression of abd-A in the ectoderm, but not the mesoderm, was not reported.
Without knowing the effect of ectopic abd-A expression in the ectoderm and
considering that the transformed pattern of twist expressing cells was only an
approximation of the normal pattern, it was not possible to rule out some role for the
ectoderm in muscle patterning. Correct patterning of adult muscle may be a product
of both autonomous and inductive signals.
Pattern formation in the fifth segment male muscle, the MOL
The mechanism determining the presence or absence of one particular muscle, the
MOL, has been extensively studied (Lawrence and Johnston, 1984, 1986; Galley
33
et.al., 1991; Taylor, 1992). The MOL is male specific; presumably its expression is
in some way regulated by both the sex-determining genes and the muscle-determining
genes. The developmental regulation of this muscle was originally investigated in
terms of a "male" induction signal versus autonomous mesodermal patterning
(Lawrence and Johnston, 1984;1985). Only more recently has the role of the specific
sex-determining genes been investigated (Gailey et al., 1991, Taylor, 1992).
Lawrence and Johnston (1984) use gynandromorphs (XX flies with clones of XO
cells, XX/ /XO flies) to locate the region on the blastoderm where cells must be male
(XO) for the male muscle to develop. This fate map, showing the relative location of
the MOL patterning information in the developing blastoderm, was constructed by
determining the probability that any two structures are of different genotype
(phenotype) are on different sides of a clonal boundary. These probabilities are then
used to create a two dimensional representation of the embryonic blastoderm showing
the relative location of the primordia that give rise to adult structures. In general, the
more often two structures are both in a clone, the closer their blastodermal primordia,
and the closer their relative position in the map.
To create XO areas within an )0( fly, lines of flies with specific mutations
(mitotic-loss-inducer, mit and paternal-loss-inducer, pal) were used. These mutations
cause loss of an X chromosome at a certain frequency, resulting in patches of XO
(male) tissue in predominantly XX (female) flies. These lines were constructed in
such a way that XO cuticle could be distinguished from XX cuticle by visible markers.
34
The presence or absence of a MOL was then scored against XX or XO phenotypes in
various tissues.
The focus for MOL patterning was found to map far away from the adult
epidermis primordium and close to both the adult muscle and the nervous system
primordia on the blastoderm fate map. In fact, the male patterning signal maps
equidistant from both the adult muscle and nervous system primordia. Since the
MOL focus does not map close to the primordium for the adult epidermis a patterning
role for the epidermis is unlikely. But, because the patterning information maps
equidistant from both the muscle and neural primordia it is not possible from these
experiments to distinguish between these two regions as possible sources for
patterning information.
These lines were constructed so that in a fraction of the flies loss of the paternal X
chromosome leaves the clones abd-B , generating abd-if clones in both the cuticle and
the musculature. The abd-B mutation causes a transformation of A6 and A7 into A5.
Lawrence and Johnston were, therefore, able to assay which tissue had to be male and
abd-B- to give expression of a MOL in transformed tissue; could underlying cuticle
induce the ectopic formation of a MOL, or was MOL formation dependent only on
muscle genotype. As in the mapping experiment, the presence or absence of the
MOL was found to be independent of cuticle genotype. However, because no
independent muscle specific marker was included it was not possible from these
experiments to determine whether the patterning signal was autonomous or came from
innervating nervous tissue.
35
A subsequent set of experiments incorporated a marker capable of marking
individual muscle cells (Lawrence and Johnston, 1986). Genetic mosaics were
constructed by injecting heterozygous nuclei into host fly embryos, both host and
donor tissue were assayed later for sex and genotype. Donor nuclei were
homozygous for the dominant mutation Miscadastral pigmentation (Mcp). This
mutation results in the transformation of A4 into A5, complete with ectopic
expression of the MOL in males. Host flies carried a temperature-sensitive succinate
dehydrogenase (sdh) allele, a mitochondrial marker. When host tissue was heated and
then stained for SDH activity the tissue remains clear. In the injected flies only donor
derived cells stain for SDH after heating. Cuticle-specific markers (yellow and
cinnabar) were used to distinguish donor from host derived cuticle.
As in the gynandromorph experiments cuticle genotype was not found to influence
MOL expression. Male muscles were seen associated with female cuticle in both A4
and A5. Similarly, male cuticle was not necessarily accompanied by the presence of
a male muscle. Unexpectedly, in some cases XX muscle cells formed a male muscle
in both A4 and AS and in other cases XY tissue failed to form the male muscle.
Further, expression of the male muscle in A4 (part of the Mcp phenotype) was
independent of the state of the Mcp gene in the muscle or cuticle of that segment.
Chromosomally male muscle was neither necessary, nor sufficient, for expression of
the male phenotype. Apparently, formation of the MOL is neither controlled by
cuticular induction nor cell-autonomous.
36
This leaves induction by the innervating tissue as the most likely candidate for the
source of patterning information for the MOL. Note that the gynandromorph fate
mapping experiment (Lawrence and Johnston, 1984) implicated autonomous or
nervous induction as equally likely mechanisms; pattern induction by the innervating
tissue was not a completely unexpected solution.
To a limited extent Lawrence and Johnston (1986) were able to examine the role
of innervating tissue in determination of the MOL. The sdh marker labels motor
neurons as well as muscle cells. Under the light microscope, however, stained
terminals are only visible when they overlay unstained (sdh+) muscle. In the cases
where this condition was met the genotype of the innervating tissue did match the
muscle pattern phenotype. Female innervating tissue corresponded to an absence of
the MOL in both A4 (Mcp- muscle) and A5. Further, in the two cases where male
muscles formed in A4 from female, Mcp+ , tissue, the innervating tissue was both
male and Mcp-. These results implicate the innervating tissue as the focus of activity
for both sex and segment specific patterning of the fifth segment MOL.
More direct evidence indicating a patterning role for the innervating tissue has
come from subsequent ablation studies (Currie and Bate, in press). Denervation of a
developing segment does not alter the pattern of non-sex-specific muscles. This is
consistent with the model developed by Bate, patterning of the non-sex-specific
abdominal muscles is not determined by induction by the nervous system. Control of
the MOL is apparently different from the type of control found in other abdominal
muscles.
37
Is this reliance on induction from innervating tissue a general feature of sexspecific muscle? In Manducca the larval muscle remnants form the normal sexspecific scaffolding even after denervation during muscle formation, but fail to recruit
new myoblasts to regrow into adult muscles(Thorn and Truman, 1989). Regrowth,
not patterning, is innervation dependent. However, this could be a consequence of
development from larval templates and Drosophila genital muscles most probably do
not form from a larval template. As yet there is no answer to this question, but
preliminary studies of flies in which the terminal nerves are ablated during pupation
shows that the genital muscles may depend on innervation for expression of some
aspects of their normal sex-specific phenotype (Taylor, personal communication).
Summary
1) Drosophila are holometabolous insects, with different and distinct sets of larval and
adult muscles.
2) In Drosophila, sex-determination is a function of the ratio of sex-chromosomes to
autosomes. In most tissue this ratio is transmitted to sex-differentiation genes by a
short cascade of sex-determining genes. In some tissues, most importantly the MOL,
a modified cascade provides this function. The genes responsible for correct
patterning of the genital muscles are unknown.
38
3) The pattern of larval muscles does not appear to be a result of epidermal or neural
induction, but rather a result of autonomous patterning. The correct final pattern
may, however, be the result of interaction between the tissues.
4) Most adult muscles develop de novo from precursor cells set aside in the embryo,
not from larval muscles.
5) As in the larva, determination of adult non-sex-specific muscles is not a function of
either epidermal or neural induction, rather it appears to be tissue autonomous.
6) Conversely, the determination of the fifth segment male muscle, the MOL, is nonautonomous and dependent on innervation by male (XO) tissue.
7) Whether determination of the sex-specific genital muscles is innervation-dependent
or independent is unknown.
39
Chapter 2.
The Role of the doublesex Gene
in the Determination of the Male
and Female Genital Muscles
of Drosophila melanogaster
Thomas J. S. Merritt
To be submitted to Developmental Biology.
40
Introduction
Adults of many species possess extensive sexual dimorphisms reflecting the
different roles each sex plays in reproduction. Development of these sexually
dimorphic structures and behavioral is a product of both tissue- and sex-specific
regulation. Analysis of the development of sexually dimorphic characteristics in
Drosophila has been aided by our extensive understanding of the genetic and
molecular regulation of sexual determination (for recent review, Burtis, 1993).
Genetic analysis has suggested that there are two output pathways that control the
sexual differentiation of somatic tissues. In one pathway, the doublesex gene
regulates peripheral development and at least one sexual difference in the central
nervous system. In the second pathway, an unknown gene, or set of genes, regulates
development of a male-specific muscle, the Muscle of Lawrence (MOL), and male
sexual behavior (Taylor, 1992; Taylor, 1994).
Although numerous sexual dimorphisms have been described in adult Drosophila,
the MOL is the only sex-specific muscle that has been extensively studied (Lawrence
and Johnston, 1984; Lawrence and Johnston, 1986; Gailey et al., 1991; Taylor, 1992;
Taylor and Knittel, in prep). A separate set of muscles, associated with the male and
female genitalia, have previously been incompletely described (Ferris, 1950; Miller,
41
1950; Crosse ly, 1978). This study examines whether or not the development of these
other sex-specific muscles, associated with the genitalia, are dependent on the
doublesex branch of the sex-determination regulatory gene cascade.
In the development of sex-specific traits of many species, an initial chromosomal
difference is translated into sexually dimorphic characteristics. In Drosophila, five
genes translate the primary sex-determining signal, the ratio of X chromosomes to
autosomes, into the signal for sexual differentiation: Sex lethal (Sxl), transformer (tra),
transformer-2 (tra-2), intersex (ix) and doublesex (dsx) (for reviews: Baker and Ridge,
1980; Baker and Belote, 1983; Baker, 1989; Slee and Bownes, 1990; Steinmann-
Zwicky et al., 1990; Belote, 1992; Burtis and Wolfner, 1992; Cline, 1993).
Sxl, tra and tra-2 act through the dsx gene to control proper female development.
Mutations in tra, tra-2 or the somatic tissue functions of Sxl cause females to develop
as somatic males; these genes act to suppress masculinization in female development.
Suppression of masculinization in XX flies also requires the product of the intersex
(ix) gene. Somatic male development, on the other hand, is unaffected by the absence
of function of these four genes.
Among these sex-determining genes dsx is unique in that a functional, sex-
specific, dsx protein is required for proper sexual differentiation of either sex (Baker
and Ridge, 1980; Baker and Wolfner, 1988; Burtis and Baker; 1989; Burtis et al.,
1991). In females the tra and tra-2 proteins work together to direct the female
specific splice of the dsx primary transcript (Baker and Wolfner, 1988; Nagoshi et
al., 1988; Burtis and Baker, 1989; Ryner and Baker, 1991; Hedley and Maniatis,
42
1991; Hoshijima et al., 1991). It has been proposed that the resulting female-specific
protein, DSr, acts to repress male-specific development (Burtis and Baker 1980,
Baker and Belote 1983) and activate female development (Taylor and Truman, 1992).
In at least one case DSX11 is thought to directly control female-specific development
by activating gene transcription (Burtis et al., 1991; Coshigano and Wensink, 1993).
In males, a functional tra protein is absent and the dsx primary transcript is spliced
into a male-specific pattern by default (Nagoshi et al., 1991). The resulting malespecific dsx protein, DSXM, acts to repress female development (Burtis and Baker,
1980, Baker and Belote, 1983) and to activate male development (McRobert and
Tompkins, 1985; Taylor and Truman, 1992; Jursnich and Burtis, 1993).
Absence of dsx function results in intersexual development of chromosomally male
or female flies (Hildreth, 1965; Baker and Ridge, 1980; Postlethwait et al., 1980; Ota
et al., 1981; Bownes and Nothiger, 1981; Nothiger et al. 1980, Chapman and
Wolfner, 1988; Feng et al., 1991). In these intersexes, sex-specific structures, such
as the genitalia, that develop from separate male and female primordia both develop.
Structures, such as the analia, that develop from a single primordium differentiate as
intermediate structures, exhibiting characteristics of both males and females. Other
structures, such as the sex-combs, develop in dsx intersexes as an average of the male
and female condition (Baker and Ridge, 1980).
Involvement on dsx in regulation of development of sex-specific traits is not,
however, universal (Mc Robert and Tompkins, 1985; Taylor, 1992; Taylor et al.,
1994). Although XX recessive homozygotes are intersexual in many traits, they do
43
not show any male courtship behavior. Similarly, XX; du') Df flies, which develop
as somatic males, show no male courtship behavior. This suggests that the CNS is
not completely transformed by du mutations (McRobert and Tompkins, 1985; Taylor
et al., 1994). Additionally, formation of the MOL is unaffected by mutations in dsx;
XX du' intersexes do not express a MOL, while XY dsx" intersexes always do
(Taylor, 1992). With the number of sex-specific characteristics that develop
independent of dsx regulation growing, I have examined the role of the dsx gene in
the development of the only other known set of sex-specific muscles, the male- and
female-specific genital muscles.
As a general rule, sex-specific structures develop in two ways: from the
differentiation of a single primordium, which adopts a male or female morphology, or
from the differentiation of one of a pair of dual primordia, each primordia with a
fixed, sex-specific fate. In Drosophila melanogaster, development of structures from
the genital disc involves both of these strategies. This imaginal disc is composed of
three distinct primordia that give rise to the analia, male genitalia and female genitalia
(Nothiger et al., 1977; Schupbach et al., 1978; Epper, 1981; 1983a; Epper and
Nothiger, 1982; Epper and Bryant, 1982). A single anal primordia adopts either a
male or female pattern, becoming either the two dorsal/ventral anal plates of the
female or the two lateral anal plates of the male (Epper and Bryant, 1982; Taylor,
1989). By contrast there are two separate genital primordia within the disc: a female
genital primordium that develops into the internal and external genitalia in females
(Epper, 1983b; Taylor, 1989) and a male genital primordia that develops into the
44
internal and external genitalia in males (Epper and Bryant, 1982; Taylor, 1989). In a
normal individual fly only one genital primordium differentiates; the other regresses
and appears to degenerate (Epper and Bryant, 1982; Epper and Nothiger, 1982;
Epper, 1983; Taylor, 1989).
Materials and Methods
Drosophila stocks
The identification of genital muscles in wildtype flies was aided by the use of a P-
element line (line 72-3, kindly supplied by Dr. S. Tobin), in which the expression of
a reporter, B-galactosidase, is driven by the promotor from a muscle-specific actin
gene, 79B actin (P[79B actin-lacZ, ly+j; Sanchez et al. 1983; Courchesne-Smith and
Tobin, 1989).
I used both recessive and dominant mutant alleles of the doublesex gene to
examine genital muscles in flies with intersexual development. For the purpose of
mapping genital muscles in mutants, I constructed a P[79B actin-lacZ, Ty+) pP dsx'
rys0'6 sr es/TM2, ry ubxl" e line (P[79B-lac2] dsx'/7312) by meiotic recombination and
standard crosses. The dsx- mutants examined in this study were either P[79B -lacZJ
dsx' homozygotes or 13179B-lacZ1 ds 1Df(3R)dsx15 transheterozygotes. The mutant
transheterozygotes were generated by crosses between X1Y;P[79B-lacZ] dsx'/TM2 and
y w/y w,Df(3R)dsx151 7316B flies and were used to control for potential background
effects. Flies that were mutant for a dominant dsx allele and had the P[79B -lacZJ
45
insert were generated by crosses between either y+ NY;dsxD Sb eT I TM6B, Tb Hu es
ca (daD/TM6B)or BrY;dseITM6B, Tb Hu es ca (dsxm/TM6B) males (provided by Drs
B. S. Baker and R. Nagoshi) and females from the 72-3 line. Descriptions of the du
and other marker alleles used are found in Lindsley and Zimm (1992).
Chromosomally male and female dsx intersexes are not distinguishable by external
phenotype; chromosomal sex was, therefore, determined by three methods. The sex
chromosome markers, yellow (y) and y+Y, were used to distinguish yly+Y;P[79BlacZ] dsx1IP[79B-lacZ1 dsx' males (normal body color) from y/y;P[79B-lacZ1
dsx11P179B-lacZ1 dsx' females (light body color). Likewise ylY;P[79B-lacZ]
dsxilDf(3R)dsx15 males were distinguished from y / +;P[79B -lacZJ dsx1IDf(3R)dsx15
females. In some early experiments the P[79B-lacZ] du 1 ITM2 line had not been
formed as a y/y+Y line; in these cases XY P[79B-lacZ]
homozygotes were sexed
by the presence of the MOL muscle and XX dui by the absence of the MOL (Taylor,
1992).
All flies were raised at room temperature on a diet of sugar, cornmeal and agar,
with propionic acid added as a mold inhibitor.
Visualization of genital muscles in wildtype and dsx mutant flies
In lines containing the P[79B-lacZ1 insert, genital muscles were visualized by
either monitoring B-galactosidase activity or by immunohistochemical labeling of the
enzyme. In either case, terminal abdominal segments from pharate (fully developed
flies that have yet to emerge from the pupal case) or adult flies were dissected to
46
expose the genital muscles. For histochemical identification, abdomens were
incubated in the X-gal reagent (0.2% 5-bromo-4-chloro-3-imdoxly-B-D-
galactopyranoside) dissolved in a reaction mixture (Asburner, 1989). Most abdomens
were incubated in X-gal for 4 hours at room temperature. Longer incubation times
did not change the pattern of stained genital muscles. After staining, abdominal
segments were fixed for about an hour in 4% paraformaldehyde in 0.1M phosphate
buffer (4% PFD in PBS) and mounted in Permount (Sigma) resin between two cover
slips.
Abdominal preparations to be labeled for B-galactosidase using
immunohistochemistry were fixed in 4% PFD in PBS for 1 to 12 hours and then
rinsed in PBS. Longer fixation times were found to reduce the level of background
staining. Abdomens were initially blocked in 10% heat inactivated normal-goat-serum
(NGS) in PBS for 1 hour and then incubated for 12 to 24 hours in anti-13-galactosidase
antisera (Cappel) at a dilution of 1:10,000 in 0.1M PBS containing 0.1% Triton-X
and 2% heat-inactivated normal goat serum (PBS-TX-NGS). Following incubation in
the primary antibody, the abdomens were incubated in a biotinilated secondary
antibody (Vectakit, Vector Laboratory) at a dilution of 1:200 in PBS-TX-NGS.
Finally, abdomens were incubated in the ABC reagent (Vector Laboratory).
Diaminobenzidine (DAB; Sigma), in the presence of 13-D-glucose and glucose oxidase
in 0.1 M Tris buffer (pH 8.2), was used as the chromogen (Metcalf, 1985). The
preparations were mounted in Permount resin between two cover slips.
47
Terminal abdominal preparations were examined using polarized light or
differential interference contrast optics to allow identification of both stained and non-
stained muscle fibers. Muscle presence and staining were scored as separate
phenotypes in both wildtype and the dsx mutant animals. Further, each side was
scored as a separate case.
Results obtained by the use of either staining technique were consistent in both
wildtype and dsx mutant flies. Antibody stained preparations were used to map the
individual muscles and their insertion points in wildtype animals, since resolution of
individual fibers and insertion points was better in these preparations. However,
poor penetration of one or more of the immunohistochemical reagents often gave
incomplete staining, especially in older animals. The analysis of muscle presence and
staining included only wildtype and mutant animals stained for X-gal to insure high
reproducability and sensitivity.
The pattern of stained muscles expands within 12 to 24 hours post eclosion to
include a number of non-sex-specific muscle fibers in other regions of the abdomen.
Segmental homologues of the MOL were found to stain in both males and females
(Taylor and Knittel, in preparation). Additionally, ventral longitudinal and ventral
transverse muscles stain in older animals. In animals stained as late as two weeks, no
additional genital muscles were stained.
48
Results
In male and female Drosophila large, multi-fiber muscles attach to the genitalia
and the terminal abdominal segments in a stereotypical, bilaterally symmetrical,
pattern (Ferris, 1950; Miller, 1950; Figure 2.1A, Table 2.1A). In males and females
from a P[79B-lacZ] line, a subset of these genital muscles expressed B-galactosidase
(Figure 2.1A and 2.1B, see Materials and Methods for details of the complete
genotype). This subset of the genital muscles stained for reporter gene expression by
midway through adult development, similar to the onset of staining in the MOL,
another sex-specific muscle (Courchesne-Smith and Tobin, 1989; Taylor and Knittel,
in prep). Early descriptions of abdominal muscles in Drosophila have only
incompletely described the number and pattern of male and female muscles associated
with the external genitalia (e.g. Ferris, 1950; Miller, 1950; Crossley, 1978); I have
used the additional information obtained from the pattern of stained muscles in the
P[79B-lacZ] line to further distinguish individual genital muscles and so to facilitate
the comparison of wildtype muscles to those in doubles& mutants. In my analysis of
genital muscles I have developed an identifying numbering system concerned only
with the musculature having insertion points on the genitalia and the posterior
abdominal segments. In cases where muscles had already been named or numbered,
for example the penis extender muscle (Ferris, 1950), I include that information in
49
our description of the muscles. No muscles solely associated with the internal
genitalia, such as muscles within the sperm pump or around the spermatatheca, are
part of this study.
Figure 2.1 Composite photomicrographs of adult wildtype female terminalia Muscles
expressing the P[79B -lacZJ reporter gene were stained using an a-B-galactosidase
antibody (see Materials and Methods). Because of the thickness of the terminalia, at
this magnification no single plane of focus shows all muscles and/or muscle
attachment sites. The examples in this figure are, therefore, composites from images
at multiple focal planes.
2.1A. Male internal terminalia All 10 of the male genital muscles, mgml-mgm10,
are visible in this example. One of each bilateral pair is labeled. The structures of
the male terminalia to which the mgms attach are: A6 tergite (6t); A6 ventral soft
cuticle (6sc); genital arch (GA); penis apparatus (PA); penis apodeme (P); and the
hypandrium (H).
2.1B. Female internal terminalia Five of the female genital muscles are visible in
this example, fgm4, fgm7, fgm8, fgm9 and fgm10. One of each bilateral pair is
labeled. The structures of the female genitalia to which fgms attach are: A7 tergite
(7t); A7 sternite (7s); A8 tergite (8t); and uterus (U). In this view, fgml, fgm2,
fgm3 and fgm5 are all hidden from view by the body of the uterus. fgm6, and its
ventral insertion of the A7 tergite, are below the plane of focus and hidden by fgm7.
in
51
Figure 2.2 Schematic drawings of adult wildtype male and female terminalia These
drawings are adapted from camera lucida drawings and have been schematisized to
more clearly demonstrate the relative positions of the genital muscles and their
attachment points.
2.2A. Male internal terminalia. On the right, each of the male genital muscles is
drawn in outline and numbered. On the left, muscles that stain for B-galactosidase in
flies carrying the 13179B-lacZI reporter gene are uniformly blacked in; muscles that
did not stain are stippled. The muscle attachment points, and other important
structures within the male terminalia, are: A6 tergite (6t) and sternite (6s); genital
arch (GA); anal plates (AN); penis apodeme (P); penis apparatus (PA); and the
hypandrium (H). The anal plates lie posterior to (behind, in this drawing) the genital
arch. All cuticular structures are drawn in a broken line.
2.2B. Female internal terminalia. As in the drawing of the male terminalia, on the
right, each of the female genital muscles is drawn in outline and numbered. On the
left, muscles that stain for B-galactosidase in flies carrying the P[79B-lacZ] reporter
gene are uniformly blacked in; muscles that did not stain are stippled. The muscle
attachment points, and other important structures within the female terminalia, are:
A7 tergite (7t) and sternite (7s); A8 tergite (8t); anal plates (AN); uterus (U); and two
ducts that enter the uterus at the joint of the oviduct (0) with the uterus, the seminal
receptacle (SR) and spermatathecae (ST).
In these drawings the anterior-posterior body axis is perpendicular to the plane of the
paper, with anterior coming toward the reader. Dor is dorsal, Ven is ventral.
Dor
. ---- \
A
t
6s
Ven
Dor
B
Figure 2.2
Ven
53
Genital muscles in wildtype male flies
Associated with the external genitalia of wildtype males are ten multifiber muscles
which I have called male genital muscles (mgms; Figure 2.1A). When visualized
using polarized light, the number, position and size of these muscles was identical in
Canton-S males and P[79B-lacZ] homozygous males (data not shown). Six of these
muscles label for B-galactosidase in the P[79B -IacZJ homozygotes (Figures 2.1A and
2.2A, Table 2.1A) and P[79B -lacZJ hemizygotes, which carry only one copy of the
reporter gene construct (data not shown). Muscle staining for P179B-lacZ1 reporter
activity was limited to skeletal muscles and was not found in muscles associated solely
with the internal genitalia, such as the sperm pump and accessory glands.
For convenience, I have divided the ten male genital muscles into three sets, based
on their anatomical location. The first set, mgml-mgm5, includes the most central
muscles - those which have both muscle attachment points on, or within, different
structures of the external male genitalia The most dorsal genital muscle, mgml,
attached to the anterior lip of the genital arch and onto the base of the penis
apparatus. Two muscles, mgm2 and mgm3, attached laterally to the genital arch and
extended ventrally to insert on the outer edge of the hypandrium. Although these
muscles are similar, mgm2 can be distinguished by its slightly more dorsal insertion
on the genital arch. The muscle recognized as the penis extender muscle by Ferris
(1950), mgm4, attached to the hypandrium and the apex of the penis apodeme. The
penis retractor (Ferris, 1950), mgm5, is the most ventral of the central set of male
54
genital muscles. It attached to the anterior (internal) ventral edge of the hypandrium
and to the base of the penis apodeme.
Four of the five muscles from the central set stained for B-galactosidase in P[79B-
lacZ1 males (Figures 2.1A and 2.2A, Table 2.1A). In all animals examined, the
muscles mgm2, mgm3 and mgm5 stained uniformly and heavily, irrespective of
whether one or two copies of the reporter gene were present. Within mgml, muscle
fibers in the more dorsal half of the muscle often stained more darkly than muscle
fibers in the more ventral half. This differential staining of mgml was more common
in pharate adults and adults with only a single copy of the P[79B-laal reporter than
in adults with two copies of the reporter gene. Under these conditions the X-gal
product was often limited to the muscle nuclei and not distributed throughout the
cytoplasm. Due to this differential staining, we denote the two regions of mgml
separately as mgmla and mgmlb. The only non-staining muscle in this central group
was the penis extender muscle, mgm4; it was never found to stain for B-galactosidase
with either X-gal or anti-B-galactosidase treatment.
Outside of this central ring were four muscles, mgm6-mgm9, which have one
attachment point on the male genitalia and the other attachment point on the sixth
(last) abdominal segment.
Dorsally, mgm6 extended from the A6 tergite to the
outside of the lip of the genital arch, opposite from the mgml insertion. The next
muscle, mgm7, has one attachment point on the genital arch, in between mgml and
mgm6, and a second attachment point on the A6 tergite. Laterally, mgm8 extended
from the A6 tergite to the genital arch near the anterior insertion of mgm3.
55
Ventrally, mgm9 inserted on the lateral soft cuticle between the A6 tergite and
sternite and extended to the anterior lip of the hypandrium, ventral to the insertion of
mgm5. Of the muscles in this second set, only the dorsal muscle, mgm7, stained for
B-galactosidase.
The last male genital muscle, mgm10, is associated only with the terminal
abdominal region. This muscle lies completely within A6, inserting dorsally on the
tergite and ventrally on the soft cuticle between the tergite and sternite. This muscle
has been included in our study, even though neither of its insertion points were on the
genitalia, because it is male-specific and, like the genital muscles in the central and
surrounding sets, stained for the P[79B -lacZJ reporter gene.
Genital muscles in wildtype female flies
A similar number of muscles have also been identified associated with the genitalia
and terminal segments of wildtype females, the female genital muscles (fgms, Figures
2.1B and 2.2B). Of these ten muscles, six muscles stained for B-galactosidase in
females of the P[79B -lacZJ line. As in the male P[79B -lacZJ flies, muscular staining
for B-galactosidase was confined to skeletal muscles with attachments on the external
genitalia and/or terminal segments; the circular muscles that surround the internal
genital structures, such as the spermatatheca, uterus and seminal receptacle were
never stained for reporter gene expression.
Similar to the male muscles, for convenience in description I divide the female
muscles into two sets based on insertion points: a central and an external set. The
56
central set is composed of four muscles, fgml - fgm4, which have attachments to
structures derived from the genital disc. Three of the muscles, fgml, fgm2 and
fgm3, attached to the A8 tergite and the uterus. The most dorsal muscle, fgml
(called muscle 148 in Miller, 1950), inserted dorsally, near the midline, on the A8
tergite and attached ventrally to the base of the uterus. More laterally, fgm2 (muscle
146; Miller, 1950) extended from the ventral part of the A8 tergite to the base of the
uterus at the midline, crossing internally over fgml. Another lateral muscle, fgm3
(muscle 147; Miller, 1950) attached to the ventral edge of the A8 tergite and extended
up, in a basket-like pattern, around the dorsal side of the uterus, ending about two
thirds of the way along the uterus. The most ventral of the central set of muscles,
fgm4, (muscle 149; Miller, 1950), extended from the base of the uterus up, along the
ventral side of the uterus, to insert at the same level as the attachment of fgm3 to the
uterus. In the central set of female genital muscles, fgml, fgm3 and fgm4, but not
fgm2, stained for B-galactosidase (Figures 2.1B and 2.2B).
The external set of female genital muscles contains six muscles, fgm5-fgm10,
which surround the female genitalia (Figures 2.1B and 2.2B). In the dorso-lateral
region, three muscles attached to both the A7 and A8 tergite have previously been
identified as a single muscle (muscle 139; Miller (1950). The most dorsal of the
three, fgm5, extends along the anterior-posterior body axis between A7 and A8.
From a more lateral position, fgm6 extends obliquely between the tergites, inserting
on A8 in the same region as fgm5. The third muscle in this group, fgm7, inserts on
the A7 tergite between fgm5 and fgm6 and extends, like fgm5, along the anterior-
57
posterior body axis, crossing fgm6, to insert more ventrally on the A8 tergite. Of
these dorso-lateral muscles only fgm6 stained for B-galactosidase in P[79B-lacZ1
females (Figures 2.1B and 2.2B).
The last three muscles of this external group each attach to the uterus. From a
lateral attachment in the A7 tergite, fgm8 extended to the body of the uterus, inserting
at the same level as the epidermal ducts of spermatatheca and seminal receptacle.
This is the longest of the female muscles and, along with fgml, the easiest to identify
in wildtype females. Near to fgm8, fgm9 extended from the A7 tergite to the base of
the uterus. Along the ventral midline, fgml0 (144; Miller, 1950) stretched from the
anterior edge of the A7 sternite to the ventral region at the base of the uterus. All
three of these muscles, fgm8, fgm9 and fgm10, stained for B-galactosidase in P[79BlacZ1 females(Figures 2.1B and 2.2B).
58
Figure 2.3 Composite photomicrographs of adult, dsx-mutant, intersexual terminalia.
Muscles expressing the P[79B -lacZ] reporter gene were stained using the X-gal
reagent (see Material and Methods). As in Figure 1, these are composite images
created from photomicrographs at multiple planes of focus.
2.3A XY; dsxildsx1 intersexual terminalia. One from each pair of genital muscles
present is labeled. The structures of the terminalia to which genital muscles attach
are: A7 tergite (7t); A7 sternite (7s); A8 tergite (8t); genital arch (GA); penis
apparatus (PA); hypandrium (H); soft cuticle of the female genitalia (in this image, a
female bristle, fb, marks one edge of this region). In this example, both of the pair
of mgmls are present and show 13179B-laal activity. A pair of mgm2/3 are also
present, but only the left (our view) muscle shows P[79B -lacZJ activity. Only one
mgml0 is present; it also shows P[79B-lacZ] activity. No other male genital muscle
is present in this example; notice the lack of muscle fibers between the hypandrium
and the penis apparatus and between the genital arch and the terminal tergite. A pair
of fgm4 are found at the mouth of the genital knob; both show P[79B -lacZ]
expression. fgml0 is present, but shows no expression of the P[79B-lacZ1 reporter
gene. A thin example of the ectopic staining muscle fibers (EM) is present between
the right A7 tergite and sternite.
In some animals, stray muscles, that could not be identified as male or female
genital muscles, were present in the terminalia. The stained muscle in the dorsal
right half of the A7 tergite is an example of such a muscle (marked with arrow). It is
possible that this particular muscle represents an fgm6 with a posterior insertion in
A7, instead of A8 (no muscle extends between A7 and A8 on this side of the animal)
but we have no way of verifying that identification. Such unidentifiable muscles were
uncommon.
2.3B XX; dsxlIdal intersexual terminalia. Muscles and muscle attachment points are
labeled as in 3A. In this example 1 mgml is present. It stains for P[79B-lacZ]
expression. Similarly, 1 mgm2/3 is present and stained. Two mgml0 are present
and show P[79B-lacZ] activity. No other male genital muscles are present in this
example. Again, notice the lack of muscle fibers between the hypandrium and penis
apparatus, between the hypandrium and the soft cuticle ventral to the hypandrium or
between the genital arch and the terminal tergite. fgm4 is present and stained for
P[79B -lacZJ expression in both sides of this animal. On the right side one fgm5/7 is
present. Though out of this plane of focus, fgml0 is present, but unstained; it lies
anterior, and internal (toward the reader) of the genital knob. No other female genital
muscle is present in this example. Notice the lack of staining fibers between the A7
tergite (the bristles on the right side of this example are on the A7 tergite) and sternite
(out of focus in this view). In addition, in this example, a spermatethecae (Sr), one
of the ducts that empties into the uterus, is present over the mouth of the genital knob
(GN). The staining in the genital knob, not associated with any muscle fibers, is nonspecific; similar staining is seen in wildtype uteri containing eggs.
59
2.3C XX;dsxM /+ intersexual terminalia
Muscles and muscle attachment points are
labeled as in 3A. In this example, mgml, mgm2/3 and mgml0 is each present and
stains for PR9B-lacZ1 activity on both sides of the animal. Small, unstained, muscles
extend between the genital arch and the A8 tergite (marked with arrow). These could
be rudimentary examples of mgm6 or mgm8. No other male genital muscles are
present is this example. One fgm6 is present, and shows P[79B -lacZJ activity, on the
left side of the animal; no fgm6 was present on the other side of the animal (out of
this view). fgm 10 is present on both sides, but unstained. No other female muscles
are present. Note the presence of the large ectopic staining muscle (EM) spanning
between the A7 tergite and sternite on both sides.
09
61
Figure 2.3
62
Figure 2.4 Schematic drawing of the adult dsx mutant terminalia.
The pattern of genital muscles was similar in both the XX and XY dsx-recessive and
XX dsx-dominant intersexes (see Table 2.1) and is represented here in a single
composite drawing. The largest difference in the terminal muscles between the
dominant and recessive mutants was in the frequency and size of the ectopic staining
muscle (EM; see Results)
On the right, each of the genital muscles is drawn in outline and numbered. On
the left, muscles that stain for 13-galactosidase in mutant flies carrying the P[79B-lacZ1
reporter gene are uniformly blacked in; muscles that did not stain are stippled. The
muscle attachment points, and other important structures within the intersexual
terminalia, are: A6 tergite (6t) and sternite (6s); A7 tergite (7t) and sternite (7s); A8
tergite (8t); genital arch (GA); anal plates (AN); penis apodeme (P); penis apparatus
(PA); and the hypandrium (H). In the majority of intersexual flies examined, a uterus
was not identifiable. Instead the female genitalia were everted, to a greater or lesser
degree depending on the individual, from the terminalia in cuticular pouch, the genital
knob (GN). The anal plates lie posterior to (behind, in this drawing) the genital arch;
the genital knob is external, posterior to the A7 sternite. All cuticular structures are
drawn in a broken line. In these drawings the anterior-posterior body axis is
perpendicular to the plane of the paper, with anterior coming toward the reader. Dor
is dorsal, Ven is ventral.
6.3
---,
Dor
/
//
..---
--
...-
I
I
1
//i /
ff/,/ GA
/2- ..--
I
1
I
1
r---- --....:Nz.,
i
\
\
\
\
\
N.
.4 ' -7\--- -=...,,\\\\\
I
i
I
i
t
I
I
AN I
i _._//
N
N
\ \ \ \\\\ \ \
\
\ ""
t
1
t
1
1
1
/
mgm10
\
i
1
...-
\_
1
-..
//--\ \.
i\ .--\\PA) mgm2/3
I
1
,...
mgml
184
I
,----1
*".1
1//
f I
.....
-... .....
... -- --.,....,..--......__---_,
-... N
// /
/
// orsoi 1
7t
-- ,....---._
--- --- -..
I
I
1
t
I
fgn25/7
/
\
u\
I k
fgml 0
I
,
7s
t_JL
- -Figure 2.4
Ven
_/
N./
64
A subset of the muscles associated with the genitalia of wildtype flies is found in
dsx mutant intersexes
To determine the role of dsx in the development of sex-specific genital muscles, I
identified the male and female muscles that developed in animals mutant for recessive
and dominant alleles of du. Chromosomally male and female dsx- mutants develop
both male and female genitalia, becoming nearly identical intersexes (Hildreth, 1965;
Baker and Ridge, 1980). I foresaw three possible ways in which male and female
genital muscles might be arranged in these mutants: 1) two complete sets of muscles,
one set associated with the male genitalia and one associated with the female genitalia;
2) only one set of muscles, which would be distributed in some fashion between male
and female genital structures; or 3) no muscles might develop at all. I examined the
genital muscles in two recessive dsx- genotypes, P[79B -lacZJ du 1 IP[79B-lacZI dal
and P[79B-lacZ1 dsx1IDf(3R)dsx15. The results obtained from XX and XY dsx-
mutants were the same (Table 2.1A and 2.1B) and will be included within a single
description.
Male genital muscles in dsx intersexes
In both XX and XY dsx' intersexes a largely complete, but distorted, set of
external male genitalia differentiate (Hildreth, 1965; Baker and Ridge, 1980). Among
the genital structures clearly identifiable are the genital arch, lateral plates and
claspers. The penis apparatus, and accompanying hypandrium are present, but
reduced; the penis apodeme is especially poorly developed (Hildreth, 1965, Baker and
Ridge, 1980).
65
Only a fraction of the normal male genital muscles are present in the dsx`
intersexes (Figure 2.1 A and 2.2A and table 2.1A). All distinctly identifiable male
muscles belong to the group of six male genital muscles that stained for Bgalactosidase in the P[79B-lacZ] line. Because the pattern of stained muscle in
wildtype flies having either one or two copies of the P179B-lacZ1 reporter gene was
consistent, I had expected the presence of stained muscles to provide positive
identification of a subset of the genital muscles in P[79B -lacZ] &xi homozygotes and
P[79B -lacZJ dsx1IDf(3R)dsx15 mutant transheterozygotes. Unexpectedly and in
contrast to the uniformity of staining between wildtype individuals, not all muscles
that were identified, by their attachment points, as B-galactosidase-positive muscles
stained for B-galactosidase in the dm( mutants. Some muscles consistency found to
stain in wildtype flies occasionally failed to stain in dsx- mutants. I have, therefore,
treated muscle presence and muscle staining as two separate phenotypes (Table 2.1A
and 2.1B). The frequency of muscle staining was very similar between P[79B-lacZ]
dsx1IDf(3R)dsx15 flies, flies with only a single copy of the P[79B -lacZJ insert, and
P179B-lacZ1 dsx'IP[79B -lacZJ dse flies, flies with two copies of the insert (Table
2.1A). It was unlikely, therefore, that our inability to detect B-galactosidaseexpressing fibers was a function of the number of reporter genes present.
Of the male muscles clearly identified in dsx- mutants, three, mgml, mgm2 and
mgm3, were in the central group of muscles, which have both attachment points
within the genitalia. In close to 93% of the cases examined from either recessive
genotype was found (Table 2.1A). In the dsx mutants, mgml was often smaller than
66
in wildtype flies and in over a third of the cases in which mgml was present that
muscle was unstained. The difference in staining separating mgmla from mgmlb was
not identifiable because of the variability in muscle staining; subsequently it was not
possible to distinguish between the these two regions in dsx- mutants (Table 2.1A).
Muscle fibers extending from the genital arch to the hypandrium, in positions
appropriate to either mgm2 or mgm3, were found in almost all of the P[79B-lacZ1
dal homozygotes and P[79B-lacZ1 dsxilDf(3R)dsx15 mutant transheterozygotes (Table
2.1A). As was the case for mgml, these muscles appear proportionally smaller than
in wildtype male genitalia and it was generally not possible to distinguish two separate
muscles, or to distinguish the more dorsal insertion of mgm2. I have, therefore,
scored only for the presence or absence of muscle fibers in this region, referring to
any muscle fibers present as mgm2/3. These mgm2/3 fibers stained for 13galactosidase in only about one half of the du" cases (Table 2.1A).
The only other identifiable male genital muscle found in these intersexes was
mgm10. In wildtype males, mgml0 spans the most terminal abdominal segment, A6,
extending ventrally from the tergite to the sternite. In contrast to wildtype males, XX
and XY dsx- intersexes have a pronounced A7 segment and A8 tergite, similar to the
segmental pattern found in wildtype females (Baker and Ridge, 1980). Though much
smaller than the A6 tergite, the A8 tergite is interposed between the dorsal male
genital structures and the A7 tergite and thus has the position similar to the A6 tergite
in wildtype males, wrapping around the genital arch and lateral plates. In 69% of the
dui homozygotes and 93% of the dsxilDf(3R)dsx15 transheterozygotes (Table 2.1A) a
67
muscle was attached dorsally to the A8 tergite and ventrally to a point below the
hypandrium; this muscle appears to correspond to mgm10 of wildtype males. No
muscle with attachment points in A6, that would correspond to mgm10, was found.
In du- intersexes, mgm10 was always found to stain (Table 2.1A).
Qualitatively there appears to be a correlation between overall muscle size and
staining in mutant animals. mgm10, the most regularly stained of the male genital
muscles, was also the largest genital muscle in these mutants. In contrast, the
unstained mgml and mgm2/3 were often smaller than their staining counterparts (data
not shown). This is not to say, however, that a large size was a prerequisite for
staining; some single fiber muscles were found to stain. Instead staining seems to
follow some degree of muscle development, with which size is often correlated.
Two of the genital muscles that label for B-galactosidase in wildtype male P[79BlacZ1 flies, mgm5 and mgm7, were never detected in dsx" mutants. This is unlikely
to be simply the lack of proper insertion points on the cuticle; the areas onto which
these muscles would be expected to attach were recognizable and other identified
muscles, such as mgml, attached near by. In wildtype males, mgm5 extended from
the ventral lip of the hypandrium to the base of the penis apodeme. The lip of the
hypandrium was particularly well formed in these dsx intersexes and, while the penis
apparatus is reduced, it too can be identified. Similarly, in wildtype males, mgm7
extended from the genital arch to the A6 tergite, however, no muscle was found in
this region of du mutant intersexes, although the genital arch was well formed.
68
Further, the insertion points for mgm7 were near those of two other muscles, mgml
and mgml0; both muscles which were found in the dsx- intersexes.
Because of the complexity and distortion of the genitalia in dsx- intersexes,
identification of the four male genital muscles that do not stain in P[79B-lacZ] males,
mgm4, mgm6, mgm8 or mgm9, was more difficult. In wildtype males the penis
extender, mgm4, attached to the penis apodeme and the hypandrium (Figures 2.1A,
2.2A). In contrast, no muscle fibers were ever found in this region of the duintersexes, though a hypandrium and penis apparatus were both recognizable in all
cases examined. In wildtype males, mgm9 also attached to the hypandrium, but no
similarly located muscles were found in the dsr mutants. The presence of the female
genitalia ventral to the hypandrium du' mutants makes it difficult to predict where
mgm9 would attach to the terminal abdominal segment, but the attachment of any
muscle to the hypandrium would be recognizable.
Two other muscles, mgm6 and mgm8, that attached to the external genitalia and
the last abdominal segment, were not found in the dsz mutants. In some cases small
dorsal groups of fibers were found to extend between the genital arch and the A8
tergite, but these were proportionally much smaller than any mgm6 found in wildtype
males. The mgm8 muscle would be expected to attach near the middle of mgm2/3
and span between the genital arch and the A8 tergite. No muscles were found to
attach to the lateral plate and the A8 tergite in this area. If mgm6 or mgm8 were
present at all, in either the P[79B -lacZJ dsxl homozygotes or the P[79B -lacZJ
dsx1IDf(3R)dsx15 mutant transheterozygotes, it was only in a greatly reduced form.
69
Occasionally, stray muscle fibers were present, irregularly attached to the male
genitalia in the dsr intersexes, but these did not attach at points that would be
expected from mgm6, mgm8 or any other genital muscle. Besides these small muscle
fibers no other ectopic, or unusual, muscle fibers were associated with the male
genitalia in dsx- intersexes.
In summary, about half of the normal number of male genital muscles were
detected in XX and XY dsx intersexes. Two of the male genital muscles, mgml and
mgm10, were clearly present at a high frequency in the dsx intersexes. Lateral
genital muscles were also represented, although it could not be determined whether
mgm2, mgm3 or both muscles were present. While staining of these muscles for Bgalactosidase in mutants with one (P[79B -lacZJ dsx1IDf(3R)dsx15 flies) or two (P[79B-
lacZJ dsx' homozygotes) of the P[79B -lacZJ construct was not as consistent as seen in
wildtype flies it was sufficient to positively identify these four muscles. Four male
genital muscles, mgm4, mgm5, mgm7 and mgm9 were never detected in dsx"
intersexes even though at least one attachment point could unambiguously be
identified. Finally, two of the male genital muscles, mgm6 and mgm8, did not
appear to be present, although it could not be completely ruled out that infrequently
appearing fibers represented these two muscle groups.
Female genital muscles in dsx intersexes
Along with the fairly well-developed set of male genitalia, XX and XY 131-79BlacZI dsx- intersexes also possess a less well-developed set of female external and
70
internal genitalia (Hildreth, 1965; Baker and Ridge, 1980). In 6% of the P[79B-lacZ1
dui homozygotes we examined, and 12% of the P[79B-lacZ] dm 1 IDf(3R)dul5 flies,
an identifiable uterus and seminal receptacle were present. In most of the remaining
dsr flies no uterus was identifiable, instead a mass of cuticle and internal tissue had
been everted from the region of the female genitalia, forming a protrusion referred to
by Hildreth (1965) as a genital knob. This knob often contained one or more
spermatethecae. Spermatetheca were also often present within the body cavity, both
in flies with and without uteri. One P[79B -lacZJ dsx11Df(3R)dsx15 fly expressed both
a genital knob and a very reduced uterus and seminal receptacle (data not shown).
Two female genital muscles, fgm4 and fgm10, were readily identified with the
female genitalia. In wildtype females, fgm4 inserts posteriorly on the base of the
uterus and anteriorly about two thirds of the way along the body of the uterus
(Figures 2.1B and 2.2B). In approximately 85% of dsx intersexes in which a uterus
was found, a muscle was found to extend anteriorly from the base of the uterus to the
body of the uterus, with attachment points appropriate for fgm4. In those cases in
which no obvious uterus formed, the internal female genital structures were generally
partially everted into a protruding knob. A muscle often extended from the mouth of
the knob, into the cavity. Based on its attachment points I have identified this muscle
as an everted fgm4. Overall, 93% of the P[79B -lacZJ d& cases and 86% of the
P[79B-lacZ1 dsxi IDf(3R)dsx15 cases expressed a fgm4 (Table 2.1B). This muscle was
found to stain in most cases examined from either du- genotype (Table 2.1B). The
only other clearly identifiable female muscle found with the female genitalia of du'
71
mutants was fgml0 gable 2.1B). As in wildtype females, this muscle inserted on the
A7 sternite and on the base of the uterus or, in those cases in which no uterus was
found, on the cuticle ventral to the genital knob. This muscle was variable in its size,
ranging from as large as in wildtype females to as small as a quarter the number of
fibers with a noticeable disruption of their regular, ordered pattern. Further, fgml0
was never found to stain for B-galactosidase in pharate, or recently emerged, adult
dsr intersexes. fgml0 did stain in some older adult cases, but only in flies old
enough for the non-sex-specific dorsal and ventral muscles to also stain for B-
galactosidase. By contrast, in wildtype females fgml0 always had early, sex-specific
expression of B-galactosidase in the P[79B -lacZJ line.
Due to the distortion of the terminalia, the reduction of the female genitalia and
the lack of internal female landmarks in the dar intersexes it was more difficult to
identify some individual female genital muscles. In the majority of cases examined,
muscles corresponding to fgm5, fgm6 or fgm7, were found dorsally, spanning
between the A7 and A8 tergites. However, in dsr intersexes three separate muscles
were rarely found in this region; on average fewer than 2 muscles were present (1.6
+/- 0.8 in P[79B-lacZ1 cluilP[79B-lacZ1 dsxl, 1.8 +/- 1.0 in P[79B -lacZJ
dsx'IDfdsx15). In a small percentage (less than 12%) of dsr cases, a stained muscle,
which would correspond to fgm6, the only dorsolateral muscle to stain for B-
galactosidase in wildtype flies, was present. In those cases where no stained muscles
was present the unstained muscles could have corresponded to either fgm5, an
unstained fgm6 or fgm7. On the basis of the presence of cases with a stained muscle,
72
I have inferred that fgm6 was present, but unstained in most elsr- intersexes. In
reporting the data in Table 2.1B I have made the assumption that fgm6 was always
present, if any muscle was found spanning the A7 and A8 tergites. At least one
muscle, putatively fgm6, was present in approximately 93% of the P[79B-laa]
cluilP179B-lacZ1 dsi cases and 81% of the P[79B-laal du-11Dfdal5 cases, but
stained in less than 12% of those cases. In most of the cases in which a stained fgm6
was present unstained fibers were also present. The presence of unstained fibers
suggests that either fgm5 and/or fgm7 were also present in the d' intersexes. It was
only possible to distinguish fgm5 from fgm7 when they were separated by a stained
fgm6. This was rarely the case. Any muscles in this region, other than fgm6, were,
therefore, scored as fgm5/7 (Table 2.1B).
Five of the ten female genital muscles were not detected in the dsx- intersexes:
fgml, fgm2, fgm3, fgm8 and fgm9. None of the dorsal three muscles in the central
group, fgml, fgm2 or fgm3, the muscles attaching to both the A8 tergite and the
uterus or genital knob, were ever identified in these intersexes, even in the 6% to
12% of cases where a uterus was present. Because, in (Liz intersexes the anal plates
are surrounded by the genital arch it is difficult to predict what form fgml would take
in these flies, but its dorsal insertion site, the A8 tergite, was always identifiable and
its ventral insertion site, the dorsal edge of the uterus, was often identifiable, but no
appropriate muscle was ever detected. The other central muscles, fgm2 and fgm3,
insert on the A8 tergite and the uterus in wildtype females, but no such muscles were
detected in dtr intersexes. No muscles were found to insert on both the A7 tergite
73
and the uterus or genital knob, as would be expected of either fgm8 or fgm9. Of the
six muscles with attachment points on the A7 or A8 tergite and the uterus in wildtype
females only fgm4 was found in the da- intersexes.
In summary, in addition to the three or four male genital muscles, four or five of
the female genital muscles found in wildtype females were found in dsx" intersexes:
fgm4, fgm10, fgm6 and either, or both, fgm5 and fgm7. Staining of these muscles
for B-galactosidase was not as consistent as in P[79B -lacZJ females. Five of the
female genital muscles were never found: fgml, fgm2, fgm3, fgm8 and fgm9.
2.1A. Muscles associated with the male genitalia.
GENOTYPE
N
Percentage of sides with muscle
mgml
mgm2/3°
mgm5,mgm7 mgml0
null alleles
P[79B -lacZJ dsx' /P[79B -lacZJ dsx'
XX 30
97(45)
98(45)
0(0)
83(100)
P179B-lacZ] dsx11P179B-lacZ] dsx'
XY 36
100(74)
90(40)
0(0)
93(100)
Df(3R)dsx15IP[79B-lacZ] dsx'
XX 32
95(39)
87(89)
0(0)
69(100)
Df(3R)dsx15IP[79B-lacZ] dsx'
XY
18
93(82)
79(55)
0(0)
93(100)
dsxml P[79B-lacZ] dsx+
XX 40
94(63)
93(51)
0(0)
100(100)
dsx'1479B-lacZ] dsx+
XX 48
94(28)
84(42)
0(0)
96(100)
dsxml P[79B-lacZ] dsx+
XY
10
100(100)
100(100)
100(100)
100(100)
dselP[79B-lacZ] dsx+
XY
10
100(100)
100(100)
100(100)
100(100)
P[79B- lacZ] /P[79B -lacZ]
XY 30
100(100)
100(100)
100(100)
100(100)
dsxmlPt79B-lacZ1 dsx'
XX 5
100(100)
100(100)
100(100)
100(100)
dsxDIP[79B-lacZ] dsx'
XX 5
100(100)
100(100)
100(100)
100(100)
dominant alleles
control genotypes
Table 2.1A. Muscles associated with the male genitalia.
GENOTYPE
Percentage of sides with muscle
mgml
mgm2/3a
mgm5,mgm7
mgml0
null alleles
P[79B -lacZJ dsx' /P[79B -lacZJ dsx'
XX 30
97(45)
98(45)
0(0)
83(100)
P[79B -lacZJ dsx111179B-lacZ1 dsx'
XY 36
100(74)
90(40)
0(0)
93(100)
Df(3R)dsx151P[79B-lacZ]dsx1
XX 32
95(39)
87(89)
0(0)
69(100)
Df(3R)dsx15IP[79B-lacZ] dsx'
XY 18
93(82)
79(55)
0(0)
93(100)
dselP[79B-lacZ] dsx+
XX 40
94(63)
93(51)
0(0)
100(100)
dsxDIP[79B-lacZ] dsx+
XX 48
94(28)
84(42)
0(0)
96(100)
dsxmlP[79B-lacZ] dsx+
XY 10
100(100)
100(100)
100(100)
100(100)
dsxDIP[79B-lacZ] dsx+
XY 10
100(100)
100(100)
100(100)
100(100)
P[79B- lacZ]1P[79B -lacZ]
XY 30
100(100)
100(100)
100(100)
100(100)
dsxmlP[79B-lacZ] dsx'
XX 5
100(100)
100(100)
100(100)
100(100)
dsxDIP[79B-lacZ] dsx'
XX 5
100(100)
100(100)
100(100)
100(100)
dominant alleles
control genotypes
Table 2.113. Muscles associated with the female genitalia.
GENOTYPE
Percentage of sides with muscle
fgml
fgm3
fgm4
fgm6b
fgm5 /7" fgm8 ,
fgm9
null alleles
fgml0
P[79B -lacZJ dsx11P[79B-lacZ1 dsx'
XX
30
0(0)
0(0)
93(97)
92(0)
53(0)
0(0)
P[79B -lacZJ dsx'IP[79B -lacZJ dsx'
100(0)
XY
36
0(0)
0(0)
94(89)
94(6)
56(0)
Df(3R)dsx15IP[79B-lacZ] dsx'
0(0)
100(0)
XX
32
0(0)
0(0)
93(80)
87(8)
63(0)
Df(3R)dsx15IP[79B-lacZ] dsx'
0(0)
100(0)
XY
18
0(0)
0(0)
78(71)
76(12)
68(0)
0(0)
100(0)
dsxmIP[79B-lacZ]
XX
40
0(0)
0(0)
65(90)
91(6)
74(0)
dsxDIP[79B-lacZ]
0(0)
100(0)
XX
48
0(0)
0(0)
89(82)
84(15)
82(0)
0(0)
100(0)
XX
30
100
100
(100)
100
(100)
100
(0)
100
(0)
(100)
dominant alleles
control genotype
P[79B-lacZIIP[79B-lacZ1
100
(100)
(100)
100
77
Genital muscles in flies expressing dominant dsx alleles
Dominant dsx alleles transform XX flies into intersexes, while XY flies develop as
normal males (Baker and Ridge, 1980; Table 2.1A). Wildtype XY flies express a
male-specific du transcript that is translated into a male-specific protein, DSXM.
Similarly, wildtype XX flies express a female-specific dsx transcript that is translated
into a female specific du protein, DSr. XX flies with a single copy of a dominant
dsx allele and a copy of a dsx+ allele generate both the male- and female-specific
transcripts (Nagoshi and Baker, 1990) and so should produce both DSXM and
DSr
proteins. Recent work on a small number of phenotypes has suggested that dsx plays
a positive role in sex-specific development; the presence of DSXM or DSX' being
required to activate development of some sex-specific characteristics (Taylor and
Truman, 1992; Coshigano and Wensink, 1993; Jursnich and Burtis, 1993). If the
presence of DSXM or DS307 was required for normal development of any of the
genital muscles I would expect dsx-dominant mutants to have muscles not seen in
recessive dsx mutants, or to produce larger or more frequent examples of muscles
already present in the null cases. I have examined the effects of two dominant dsx
alleles, dsx-Dominant (dsx") and dsx-Masculinizer (dsx"`), on the genital musculature
(Figure 2.3C; Table 2.1).
Male genital muscles in dsxD°' intersexes
The same subset of mgms found in recessive mutant intersexes, mgml, mgm2/3
and mgm10, was present in the XX;dsx-dominant intersexes. The frequency of
78
occurrence of these muscles was also similar to that seen in the recessive genotypes
(Table 2.1A). For example, mgml was present in 93-100% of du' animals and 94100% of the XX;dsxn°19P[78B-lacZ1 dsx+ animals. In these XX; d..tx-dominant
intersexes, mgm2 and mgm3 were not distinct muscles, having the same appearance
as in the du' intersexes. Again, the frequency with which mgm2/3 formed was
similar in XX,clammIP[78B-laa] dsx+ animals was similar to that with which it
formed in XX and XY dsx" intersexes. In the XX;dst°M /P[78B -lacZJ dse flies
mgml0 was found in A8, as it was in the du" intersexes. Further, the six mgms not
found in the recessive du intersexes, mgm5 and mgm7, were likewise missing from
the XX;dsx- dominant intersexes (Table 2.1A).
As in the dsx' intersexes, muscles that could be identified by their attachment
points as muscles that expressed the P[79B -lacZJ reporter in wildtype flies did not
always stain in XX;dsem/P/78B-/acZ/ d.sx+ animals.
mgml0 stained in essentially
all cases of either dominant genotype. Approximately one half of the mgm2/3's were
found to stain. The fraction of mgm2/3 and mgml0 that stained in XX;dsx-dominant
animals was similar to that found in the recessive dsx mutants (Table 2.1A). mgml
stained in almost two thirds of the XX;dselP[79B-lacZ1 cases examined and just less
than a third of the XX cluDIP179B-lacZ1 cases. It is worth noting that, in general,
XX dam1P179B-laal intersexes show a more male-like phenotype than XX
delP179B-lacZ1 intersexes (i.e. better developed external male genitalia and more
poorly developed female genitalia, Baker and Ridge, 1980). The higher frequency of
79
staining of male genital muscles in XX delP[79B-laal intersexes than in XX
delP[79B-lacZ] intersexes may be a reflection of this.
XX flies that carry a dominant dsx allele over a dsx recessive allele or a dsx
deficiency (for example: dre/dsxi or dre/dsx1) develop as somatic males,
pseudomales (Baker and Ridge, 1980). The XX; dsx"/P[79B -lacZJ dal and XX;
dsxDIPP9B-lacZ] dui pseudomales expressed a set of genital muscles identical in
number, morphology and staining to that found in XY P[79B-lacZ1 homozygotes.
The normal complement of male muscles was also present in dselDf(3R)dsx1.5 flies,
though these flies lacked the insert and could not be scored for B-galactosidase
staining. No fgm was ever detected in the dsx-dominant/dsr pseudomales.
Female genital muscles in dsxD('` intersexes
The muscles found associated with the female genitalia were also very similar in
the dominant and recessive mutant intersexes (Table 2.1B). fgm4 was found in the
majority of the cases examined and stained in over 70% of those cases in which it
was found. fgml0 was found in all cases, but was never stained in pharate and
recently eclosed flies. As with the dsx- intersexes, I have made the assumption that
fgm6 was always expressed in cases where muscles were present between the A7 and
A8 tergites. fgm6 was found to stain in 6% of the dsx"` intersexes and 12% of the
de intersexes. Additional muscles in this dorsal region were identified as fgm5/7;
these were found in 74% of the dse cases and 82% of the dsxD cases. No other fgm
could be detected in the dap' intersexes, though, as in the dsx intersexes, the
80
insertion points for fgml, fgm2, fgm3, fgm8 and fgm9 could be identified.
Ectopic muscles in dsx- dominant intersexes
The majority of the genital muscles expressed in the dsx and dominant dsx
intersexes were the same, however, one unusual muscle was found much more often
in dominant than in recessive dsx mutants, a possible case of activation by DSXM. A
muscle that extended from the A7 tergite to the A7 sternite was found in less than
15% of XX and XY dsx- intersexes (Figure 2.3A). A similar muscle was present in
72% of XX;dsxDIP[79B-laal cases and 85% of the XX;dseIP[79B-lacZJ cases
(Figure 2.3C). In general, this muscle was much larger in de animals than in du'
animals. When present in either dsx'" or dsx- animals this muscle stained for Bgalactosidase in pharate adults. Thin muscle fibers do extend between the tergite and
sternite of every segment in both male and female wildtype flies, but these muscles
have been found to stain only in flies older than 24 hours post-eclosion. It is
important to point out that this staining muscle was in A7, entirely separate from the
genitalia, this was not a genital muscle, but an masculinization of a segment not found
in wildtype males.
81
Discussion
Muscles associated with the male and female Eenilalia and terminal segments
I have described two sets of sex-specific muscles that attached to the genitalia and
terminal abdominal segments of adult flies (Figures 2.1A, 2.1B, 2.2A, 2.2B). A
subset of both male and female muscles expressed a reporter gene construct driven by
the 79B actin gene promotor (P[79B- lacZJ) by mid-pupation. Among abdominal
muscles only the MOL also shows this early pattern of reporter gene expression
(unpublished observations; Taylor and Knittel, in prep; Currie and Bate, in press).
Sex-specific expression of the P[79B -lacZ] reporter was, then, confined to
morphologically distinct, sex-specific, muscles in the abdomen. Interestingly, I find
that both males and females had a similar number of genital muscles, with a similar
fraction of those muscles showing P[79B -lacZ] activity. These similarities in muscles
and staining pattern are in contrast to the dramatically different external and internal
male and female genitalia that develop from discrete and separate primordia within the
genital disc ( Nothiger et al., 1977; Schupbach et al., 1978; Epper, 1981; Epper and
Nothiger, 1982; Epper, 1983a; Epper and Bryant, 1983). Given the difference in
external and internal genitalia, there was no reason, a priori, to expect this degree of
similarity between the male and female genital muscles. In fact, given the difference
in potential insertion points, it seemed more likely that the genitalia would have had
notably different numbers of attaching muscles.
82
With the external differences in the genitalia there was a noticeable morphological
similarity between some of the male and female genital muscles. The best example of
this similarity was between fgm5, fgm6 and fgm7, linking A7 and AS in females, and
mgm6, mgm7 and mgm8, linking A6 and the genital arch in males. These are dorsal
muscles, all from the set of external muscles that surround the genitalia in either sex.
In either group, only the central muscle stains for reporter gene activity in the P[79B-
laal line. The outer two muscles do not stain and, to a large extent, resemble
slightly modified versions of the non-sex-specific longitudinal muscles found in every
abdominal segment. Based on their similar morphologies and patterns of staining for
reporter gene activity these six muscles appear to be segmental homologues. The
dorsal most male and female muscles, mgml and fgml, are also similar, and possibly
homologous, both inserting dorsally to the genitalia, anterior to the anal plates, and
ventrally to parts of the internal genitalia. The similar number, pattern of staining for
a muscle specific reporter gene and, at least in some cases, morphology, suggests
that, unlike the rest of the external and internal genitalia, the male and female genital
muscles may develop from a single primordium.
Where might this muscle primordium be? The muscles in the adult abdominal
segments develop from imaginal cells associated with the abdominal segmental nerves
in the larvae (Bate et al., 1991; Currie and Bate; 1991). The genital muscles
precursors could be similarly associated with the terminal nerve. However,
development of the genitalia is more like development of thoracic structures, such as
the wing, than it is like development of the rest of the abdomens, since both the wing
83
and the genitalia develop from imaginal discs. Thoracic muscle precursors are
located within the thoracic imaginal discs (Lawrence, 1982; Bate, et al., 1991). The
genital muscle precursors might, similarly, be located within the genital disc. In fact,
when male and female genital discs were labeled with an antibody specific for muscle
precursor cells, a large number of cells, associated with both the male and female
genital primordia, do show specific staining (see chapter 3). While many of these
cells probably give rise to the visceral muscles associated with the internal genitalia,
the location of other cells over areas of the disc epithelium that will develop into
external structures make those cells strong candidates for the genital muscle
precursors.
The development of the genital muscles is dependent of dsx activity
A primary finding of this study was that du does have a role in the development
of sex-specific muscles. Previous work had showed that the MOL develops
independent of dsx control (Taylor, 1992), leading us to wonder if all sex-specific
abdominal muscles develop independent of dsx. We find that in dsx loss-of-function
mutations, which result in the development of both male and female genital structures,
many of the appropriate genital muscles also form (Figures 2.3A, 2.3B; Table 2.1).
Further, in concert with finding similar cuticular structures, in XX and XY dsx null
mutants muscle development is also similar in pattern and reporter expression. While
it was not possible, a priori, to predict what effect, if any, dsx would have on
expression of these muscles, only if dsx did have a role in regulating their
84
development would XX and XY dsx mutants express similar genital muscles. I
conclude, therefore, that dsx does have a role in the development of these genital
muscles and that dsx- independence is not a feature common to muscle development.
The only phenotype, other than the MOL, that has been shown to develop
independently of dsx activity is male courtship behavior (Mc Robert and Thompkins,
1985; Taylor et al., 1994). Courtship behavior is presumably controlled by the CNS.
Interestingly, development of the MOL is also controlled by innervating tissue
(Lawrence and Johnston, 1984; 1986; Currie and Bate, in press). dsx-independence
and some role of the CNS may be linked, but this possibility should be taken
cautiously as expression of at least one sex-specific CNS phenotype does require dsx
activity (Taylor and Truman, 1992). It is not yet known whether innervation has any
role in development of the genital muscles, but in light of the dependence on proper
dsx activity, it is interesting to speculate that it will be less than in the MOL. I
started this project asking whether dsx-independence was a general feature of sexspecific muscle development, instead, dsx-independence or dependence may be more a
function of the role of the CNS in the development of a phenotype.
A subset of male and female genital muscles form in dsx intersexes
To understand how the dsx gene was involved in the control of the development of
the male and female genital muscles I examined the effect of dsx loss-of-function
alleles on the pattern of adult genital muscles. I postulated three possible ways in
which muscles might be distributed between the male and female genitalia found in
85
dsx- intersexes: 1) two complete sets of muscles; 2) one set of muscles, distributed
between the male and female genitalia; or, 3)no muscles at all. I can rule out the
third possibility immediately, distinction between the first two requires closer
examination. If both sets of muscle had developed then 20 muscles would have been
expected, compared to about ten muscles if only one set had developed. Based on
insertion points and reporter gene staining characteristics, between 3 and 6 male
genital muscles and 4 or 5 female genital muscles were present in any of the dsx
intersex cases we examined, strongly suggesting that only the equivalent of one set of
genital muscles formed in the mutants (Figures 2.3A, 2.3B). In contrast to the
muscles in wildtype flies, the individual genital muscles in these intersexes were not
consistently present or stained (Table 2.1).
The regularity of formation and
frequency of staining appeared to be a characteristic of the individual muscles, not
dependent on the degree of development of particular mutant flies. For example,
mgml was found in over 90% of all cases examined, whereas fgrn5/fgm7 was found
in between 50% and 80% of the cases examined. Other muscles were never detected;
for example mgm7 in males and fgm3 in females.
The failure of certain muscles to form could result from either the absence of the
necessary muscle insertion points, or too few muscle precursor cells. I favor the
second possibility. Any distortion in the pattern of muscles present could be a result
of a distortion in the cuticle to which the muscle attaches. However, in many cases
in which the cuticle appears to be a suitable substrate for muscle attachment no
muscle forms. For example, although mgm7 was never found in the dsx intersexes,
86
its prospective insertion points on the genital arch and terminal tergite were both
identifiable, and nearby points were used by other muscles. While I cannot rule out
some defect in the cuticle, making it an unsuitable attachment site, as the cause of the
failure of certain muscles to form, the nearby insertion of other muscles makes this a
less likely possibility.
The similarity in number and pattern of genital muscles in wildtype flies (Figures
2.1A, 2.1B, 2.2A, 2.2B) already suggests that these muscles develop from a single
group of precursors. If the reduction in the number of male and female genital
muscles expressed in the dsx intersexes was a result of a limited number of available
myoblasts I would expect the total number of muscles in the intersexes to be similar
to that found in the wildtype flies. I fmd distinct examples of only two of the mgms,
mgml and mgm10. Muscles were also present in the intersexes, that could have
corresponded to either, or both, mgm2 and mgm3. Three muscles, mgm4, mgm5 and
mgm7, were never found in the intersexes. The remaining male genital muscles,
mgm6, mgm8 and mgm9, were never clearly identified, either they were not present,
or were expressed in a very reduced form that was not easily detected. In total, I
fmd three or four distinct male genital muscles, depending on whether both mgm2 and
mgm3 were present, with the remote possibility of 3 rudimentary muscles. Three of
the female genital muscles were identified in the dsx- and de"n/+ intersexes, fgm 4,
fgm6 and fgm10. Other muscles in the intersexes appear to correspond to either fgm
5 or fgm7 or both. In all I find 4 or 5 of the female genital muscles. Summing the
male and female muscles gives a minimum estimate of 7 genital muscles in the dsx
87
intersexes, and including both possible muscles from groups that I cannot distinguish
raises this number to 9, including the male muscles possibly expressed in a reduced
form raises the maximum number of muscles to 12, out of a possible 20 genital
muscles. This is consistent a reduction in the number of muscles present due to a
limiting number of myoblasts.
It may seem problematic that certain muscles always fail to develop while others
form in almost all cases; limited resources might be expected to lead to random loss,
not regular loss, of individual muscles. Why do certain muscles appear to develop at
the expense of others? It is possible that certain of the genital muscles preferentially
receive myoblasts, at the expense of nearby muscles, resulting in the consistent
appearance of some muscles and absence of others in this case of limited resources.
This could reflect a developmental bias, with earlier developing muscles reducing the
number of myoblast available for later developing muscles or could result from
certain muscles being able to actively recruit myoblasts at the expense of others. A
developmental bias does seem to be involved in control of development of the MOL
and the non-sex-specific longitudinal muscles that surround it (Taylor and Knittel, in
prep). When developing muscles were challenged by artificially limiting the number
of myoblasts available, the MOL was found to always do better than the adjacent
muscles at acquiring myoblasts. In counting the number of nuclei in the resulting
MOL and surrounding longitudinal muscles, it was found that by some means the
MOL always received a greater percentage of myoblasts than other nearby muscles
(Taylor and Knittle, in prep).
88
Along with a reduction in the total number of muscles associated with the genitalia
I were surprised to find that muscle presence was not always accompanied by muscle
staining (Table 2.1). This lack of staining could simply have been a result of too few
nuclei expressing the P[79B -lacZJ product for us to detect. In many cases, however,
single fibers, with a small number of nuclei, stained using both X-gal and the anti-B-
galactosidase antibody. Further, in other cases, large muscles, left to stain for long
periods of time (up to 24 hrs), failed to show any staining. It seems unlikely, then,
that the lack of staining simply results from an insufficient number of muscle nuclei.
Instead, if we regard staining as a separate phenotype from muscle expression, lack of
staining may result from an inability of the muscle to completely differentiate into its
sex-specific phenotype. This implies that at least one aspect of muscle phenotype,
P[79B-lac71 activity and therefore presumably 79B-actin expression, is more sensitive
to dsx activity that other aspects,such as muscle position and growth, of the muscle.
A possible positive role for DSXM
In most sex-specific phenotypes that have been studied the male or female dsx
proteins act as negative regulators, turning off the expression of inappropriate sexual
characteristics. The presence of the male dsx protein, or the female dsx protein, has
been shown to be required for the development (not repression of development) of a
few sexually dimorphic phenotypes. For example, the yolk proteins, normally
produced only in females, require the presence of DSX' for normal levels of
expression (Coshigano and Wensink, 1993). Similarly, the male-specific division
89
pattern of a set of abdominal neuroblasts requires the presence of DSXM (Taylor and
Truman, 1992). In either of these cases removal of the dsx gene activity causes a loss
of the phenotype which can be restored genetically by the return of the appropriate
dsx protein.
It is possible that the missing genital muscles, or the reduction in frequency of
staining in the muscles identified, resulted from the lack of DSX', or DSr, in flies
expressing dsx loss-of-function alleles. To test whether the presence of either dsx
protein was required for development of any of the genital muscles I examined XX;
P[79B -lacZJ dsx'1+ flies. If the normal development of any of the genital muscles
required either DSXM or DSX' I would expect to see an increase in the number of
male muscles present, or possibly an increase in the frequency of staining compared
to either XX or XY du' intersexes. However, I find no significant increase in the
total number of identifiable genital muscles present, the frequency with which any
muscle was found, nor the frequency with which a male muscle stained for the
reporter, between the dominant and recessive intersexes (Table 2.1).
There is, however, one muscle that is found in approximately 65% more dsxdominant cases than dsx-recessive cases. This was the large, staining muscle
extending between the A7 tergite and A7 sternite in over 70 percent of the dsx-
dominant intersexes examined (Figure 2.3a, 2.3C). This muscle was present in less
than 15% of the dsx-intersexes and was generally smaller than when found in the dsx-
dominant intersexes. This muscle appears to be ectopic since no such stained muscle
90
is found in wildtype male or female flies, though a band of small, non-staining muscle
fibers does extend from tergite to sternite in every abdominal segment of both sexes.
The position of this muscle was strikingly similar to that of mgm10, the lateral
staining muscle in A6 of wildtype males and A8 of both &- dominant and dsr
intersexes. A7 is not present in wildtype males, but is in females and dsx mutant
intersexes. I interpret this A7 muscle as the ectopic expression of a male phenotype
in these dsx- dominant intersexes. While expression of mgml0 itself does not require
DSXM expression (see above), the presence of DSXM appears to transform a non-sex-
specific muscle into a male phenotype. Ectopic expression of DSXM has been shown
to result in expression of sex-comb-like bristles on all of the legs (Jursnich and Burns,
1993); this may be a similar ectopic activation of a male phenotype.
91
Chapter 3.
Identification of Putative
Genital Muscle Precursor Cells
in Wildtype and dsx-Dominant
Genital Discs
Thomas J. S. Merritt
92
Introduction
During pupation the largely non-sex-specific Drosophila larvae metamorphose into
sexually dimorphic adults. Metamorphosis involves drastic changes throughout the
body of the developing fly, including changes in the epidermis, the nervous system
and the musculature. This overall change is accomplished through both
rearrangement of larval tissues and creation of adult structures de novo from imaginal
cells. In the last chapter I described a set of sex-specific muscles associated with the
adult genitalia, here I address a possible source of those muscles.
In general, the muscles present in adult insects develop either through a
rearrangement of larval muscles and subsequent fusion of myoblasts, or through
development of the muscles entirely from cells set aside earlier (reviewed in Niiesh,
1985; Bate, 1993). In the only previously studied case of development of insect
genital muscle, the genital muscles of Manducca sexta were shown to develop around
a patterning template of larval muscle remnants (Thorn and Truman, 1989).
However, this type of muscle development is rare in Drosophila; adult muscles
generally develop entirely from cells set aside in the embryo (Lawrence, 1982;
Lawrence and Brower, 1982; Bate et al., 1991). The only adult muscle in Drosophila
whose development is known to involve a larval template is a set of thoracic indirect
93
flight muscles, these muscles form around a scaffolding of partially histolysed larval
muscle remnants (Fernandes et al., 1991). Recently, however, the development of
these adult muscles has been demonstrated to be independent of the presence of these
larval muscle remnants, at least at the level of gross muscle patterning (Fernandes,
personal communication).
The muscle precursors, myoblasts, of adult muscles in Drosophila express the twist
protein (Bate et.al., 1991), a protein which is initially expressed in all presumptive
mesoderm (Thisse et.al., 1988), until midway in adult development. A decline in
twist expression by these cells coincides with the fusion of myoblasts into myotubes,
followed by muscle differentiation, which can be measured by the onset of
transcription of muscle-specific genes (Currie and Bate, 1991). For the flight and leg
muscles in the thorax, muscle precursors are found associated with larval imaginal
discs (Lawrence, 1982; Bate et.al., 1991). In abdominal segments Al through A7 the
muscle precursors are not associated with the precursors of the adult epithelium, but
occur as four separate groups of cells located at a ventral, a dorsal and two lateral
sites (Bate et.al., 1991). While adepithelial cells, presumed myoblasts, have been
located in the genital disc, their relationship to particular genital muscles has not been
described.
The genitalia, like thoracic structures, develop from an imaginal disc, the genital
disc. Gynandromorph and somatic recombination studies ( Nothiger et al., 1977;
Schupbach et al., 1978; Epper and Nothiger, 1982) as well as metamorphosis of disc
fragments (Epper, 1981; Epper, 1983a; Epper and Bryant, 1983), have shown that the
94
single imaginal disc contains three separate primordia: a male primordium, a female
primordium and an anal primordium. All three primordia are identifiable in discs
from third instar larvae of either sex. Sex specific cell growth and movement during
metamorphosis results in the final sex-specific phenotypes (Epper, 1983b; Taylor,
1989a). The male primordium is located in the anterior to lateral region of the disc,
in males this region differentiates into the set of internal and external structures of the
male reproductive system (Epper and Nothiger, 1982; Taylor, 1989a). In females the
cells of this region divide very slowly during the larval stage and eventually die
during metamorphosis (Epper and Nothiger, 1982; Epper, 1983b; Taylor, 1989a).
The female primordium occupies the ventral region of the disc, in females it develops
into the external and internal genitalia; in males this region degenerates (Epper and
Nothiger, 1982; Epper, 1983b; Taylor, 1989a). The single anal primordium is
located in the posterior-most region of the disc. In females this region gives rise to a
dorsal and a ventral anal plate. In males this same region, or a subset of this region,
gives rise to a pair of lateral anal plates (Epper and Bryant, 1982; Taylor, 1989a).
The precursors for the genital muscles could either be associated with the genital
disc or invade from the terminal abdominal segment, or both, as these sources of
myoblasts may not be mutually exclusive. In this chapter I will describe two sets of
twist-expressing cells in the discs of third instar larvae: one set associated with the
male primordia and a second set associated with the female primordia. I propose that
these sets are likely candidates for the precursor cells of at least some of the genital
muscles. Interestingly, I find no twist-expressing cells associated with the anal
95
primordia. In the last chapter the development of the genital muscles was shown to
be under the control of the doublesex gene. To further investigate the action of dsx in
the development of these muscles I have examined the twist-expressing cells in third
instar discs from flies expressing dsx-dominant mutations.
Materials and Methods:
Drosophila stocks
Genital discs were examined from larvae from fly lines used in the previous study
of adult genital muscle expression. The P-element line, P[79B actin-lacZ, rt./ was
used as the wildtype (line 72-3, kindly supplied by Dr. S. Tobin). No difference was
found in the pattern of twist-expressing cells between discs removed from flies from
line 72-3 and the few discs examined from Canton-S flies.
I used dominant mutant alleles of the doublesex gene to examine twist-expressing
cells in the genital discs of larvae with intersexual development. Larvae that were
mutant for a dsx-dominant allele were generated by crosses between either y+
IfY;dve Sb es/7316B, Tb Hu
ca (dst/TAI6B) or BrY;dsxml7M6B, Tb Hu e ca
(dsxm/TM6B) males (provided by Drs. B.S.Baker and R.Nagoshi) and females from
the 72-3 line. XX; ds.e and XX; dsxm heterozygotes were Tb+ and .13'+.
Descriptions of the dsx and other visible marker alleles used appears in Lindsley and
Zimm (1992).
96
Visualization of twist-expressing cells in genital discs
Cells expressing the twist protein were visualized using a anti-twist antibody (Dr.
B. Paterson). Discs from staged animals were dissected away from the rest of the
abdomen and fixed in 4% paraformaldehyde (4% PFD) in PBS for 1 hour and then
rinsed in PBS. Discs were blocked in 10% heat inactivated normal-goat-serum (NGS)
in PBS for 1 hour and incubated for 24 hours in anti-twist antisera at a dilution of
1:5,000 in 0.1M PBS containing 0.1% Triton-X and 2% heat-inactivated normal goat
serum (PBS-TX-NGS). Following the primary antibody incubation, the abdomens
were incubated in a biotinilated secondary antibody (Vectakit, Vector Laboratory) at a
dilution of 1:200 in PBS-TX-NGS. Finally, abdomens were incubated in ABC
(Vector Laboratory).
Diaminobenzidine (DAB; Sigma), in the presence of B-D-
glucose and glucose oxidase in 0.1 M Tris buffer (pH 8.2), was used as the
chromogen (Metcalf, 1985). The discs were mounted in Permount resin between two
cover slips. In early experiments the discs were left attached to the larval carcass,
but removal from the carcass was found to improve staining and resolution of the
twist-positive myoblasts, especially those associated with the female primordia.
97
Figure 3.1 Photomicrograph of genital discs from third instar larvae. Presumptive
myoblasts were stained using an a-twist antibody (see Materials and Methods).
3.1A XY; d.u+ third instar genital disc. twist-expressing cells are found associated
with the male genital primordia (MP), the repressed female genital primordia (RFP)
and the connection of the genital disc to the CNS (marked with an open star). Note,
especially, the twist-expressing cells associated with the region of the male primordia
that will develop into the genital arch (GA) and lateral plate (LP).
third instar genital disc. twist-expressing cells are found associated
with the female genital primordia (FP), the repressed male genital primordia (RMP)
and the connection of the genital disc to the CNS (marked with an open star). Note
the lighter staining for the a-twist antibody in the cells associated with the female
primordia.
3.1B 3,13C; dsx+
3.1C XX; dsxDom/dsx+ third instar genital disc. twist-expressing cells are found
associated with the female genital primordia (FP), the male genital primordia (MP)
and the connection of the genital disc to the CNS (marked with an open star).
98,
Figure 3.1
99
Figure 3.2 Photomicrograph of genital discs from white pre-pupae. As in Figure 3.1,
presumptive myoblasts were stained using an a-twist antibody (see Materials and
Methods).
3.2A XY; du+ white pre-pupae genital disc. The segregation of twist-expressing cells
into two separate groups is less apparent than in the younger discs (Figure 3.1). Cells
expressing the twist protein are still associated with the male and repressed female,
genital primordia, and connection of the disc to the CNS (marked with star), but have
begun to spread out across the disc epithelium.
3.2B XX; &x' white pre-pupae genital disc. As in the male disc, the twistexpressing cells have begun to spread across the disc epithelium and blur the edges of
the two groups of cells seen in the third instar discs. The repressed male primordium
has shrunken to a thin band across the upper (in this view) edge of the disc, note that
twist-expressing cells still fill this area.
100
Figure 3.2
101
Results
fwist-expressing cells are found associated with the genital disc of wildtype larvae
I examined discs from both male and female wildtype wandering third instar larvae.
The wandering third instar stage is the period during the last larval stage when
animals have been committed to enter metamorphosis. Given the association of the
twist-expressing muscle precursor cells and imaginal discs in the thorax, I had
expected to find a similar association of twist-expressing cells and the genital imaginal
disc. The genital disc, however, is unique in its organization from other imaginal
discs, in containing both active and repressed primordia. Since in each genital disc
there is a repressed genital primordium, epithelial cells which divide more slowly and
an actively dividing, unrepressed primordia, it was possible that the differences in the
myoblast population would reflect this differential potential within a given genital
disc. In addition, each genital disc has an active anal primordium that might have
associated myoblasts.
I found twist-expressing cells to be associated with both genital primordia in discs
of either sex, but never with the anal primordia (Figure 3.1A). In the genital discs
from XY larvae, a large number of twist-expressing cells were found along the
anterior and lateral regions internal to the epithelium of the male primordium.
Additionally, another large and distinct group of twist-expressing cells were also
present around the ventral region of the repressed female primordium of the disc.
twist-expressing cells were also present along the lateral projections of the disc that
102
attach it to the CNS, it was not possible to tell if these groups of cells were
continuous with the male of the female groups, or both, or neither. One feature of
the antibody labeling was that myoblasts associated with the female primordium were
more lightly labeled with the a twist antibody than were the myoblasts in the male
primordium. No twist-expressing cells were found in the region of the anal
primordia.
In genital discs from XX larvae, two populations of twist-expressing cells were
also present (Figure 3.1B). As in XY genital discs, one group of twist-expressing
cells was located in the anterior and lateral regions of the male primordia and the
other group in the ventral part of the female primordia. twist-expressing cells were
also found along the connection of the XX genital disc to the CNS. As was found for
the XY discs, myoblasts associated with the female primordium labeled more faintly
than those associated with the male primordia. No twist-expressing cells were found
associated with the anal primordia.
In addition to discs from wandering third instar larvae, I also examined a limited
number of discs from white pre-pupal males and females (Figure 3.2). At this stage
of development, larvae have just begun transformation into pupae. In both male and
female genital discs of this stage, the separation of the male and female groups of
twist-expressing cells seen in the wandering third instar discs, was no longer present
as the myoblasts began migrating to form a near continuous sheet across the
epithelium of the disc.
103
fwist-expressing cells are found associated with the genital disc of larvae
expressing dsx-dominant mutations
Females heterozygous for a dsx-dominant mutation and a wildtype allele
(dsx'/+) develop as intersexes expressing both male and female genitalia, but only
one half of the genital muscles seen in wildtype flies (see Chapter 2). Examination of
the genital discs from these mutant larvae revealed that expression of both male and
female genitalia resulted from a failure to repress the development of the male genital
primordia; the discs from third instar XX;d&)OM /+ larvae show a well developed
male, as well as, female primordia (Epper, 1981). The reason for the reduction in
the number of muscles is unknown, but could be either a result of distortion of
cuticular muscles insertion points or a limited number of myoblasts available for
formation of the genital muscles (see Chapter 2).
My first step in investigating the effect of du on the myoblasts that will form the
genital muscles was to characterize the twist-expressing cells in the genital discs of
XX;dsxD°m1+ heterozygotes (Figure 3.1C). twist-expressing myoblasts, were
associated with both the male and female primordia of these discs, in the same region
as in the wildtype discs, but not with the anal primordia. From a qualitative analysis
it does not appear that the numbers of myoblasts were grossly different between the
dsx-dominant genital discs and wildtype XY and XX discs. Staining of the myoblasts
associated with the female primordium was lighter than those associated with the male
primordium, as found in the XX and XY wildtype discs.
104
Discussion
Pattern of twist-expressing cells in genital discs from wildtype male and female
diam
In discs from XY and XX third instar larvae, labeling for the twist protein reveals
a group of cells associated with the male genital primordium and a second, more
faintly stained group associated with the female primordium (Figures 3.1A, 3.1B).
Not all of these cells are likely to be the precursor myoblasts for the male and female
genital muscles, the mgms and fgms, characterized in the last chapter. The male and
female primordia develop into the internal genitalia as well as the external genitalia.
Many of the twist-expressing cells found in these discs probably contribute to the
smaller, visceral, muscles that surround and attach exclusively to the internal
genitalia. However, some of the twist-expressing cells are located over areas of the
disc epithelium known to develop into the external genitalia (see Taylor, 1989a) and
are therefore likely candidates for the precursors of the mgms and fgms.
The presence of two discrete groups of myoblasts in the larval genital disc at first
seems to argue against the proposal that the genital muscles develop from a single
group of cells. If the twist-expressing cells found in the genital disc are the precursor
myoblasts for the genital muscles and act, effectively, as a single primordium,
myoblasts from both groups would have to persist and act as a unit to form the mgms
from a male genital disc and the fgms from a female genital disc. In fact, by the
white pre-pupal stage (the start of puparation) the two groups have merged, forming
one continuous group of myoblasts across the disc epithelium (Figure 3.2). This does
105
not prove that the myoblast do actually all persist, it is still possible that the
myoblasts associated with the repressed primordium will degenerate at a later time,
but, qualitatively, this is consistent with the myoblasts in the genital disc acting as a
single unit.
A quantitative argument for or against a single primordium could be made by
comparing the number of myoblasts within a primordium in male and female genital
discs. If muscles develop from a single primordium, genital discs from either sex
would be expected to contain a similar number of precursor myoblasts. Further, a
similar number of twist-expressing cells should be found in either primordia,
irrespective of the sex of the larvae. That is, the male primordium should have a
similar number of twist-expressing cells associated with it whether the disc was from
an XX or XY larvae; a similar case can be made for the myoblasts associated with the
female primordium.
One other important source of myoblasts needs to be considered as it is possible
that only a fraction of the mgms and fgms derive from myoblasts associated with the
genital disc: the pools of myoblasts found in every abdominal segment (Bate et al.,
1991). The lateral muscle in males, mgm10, does not insert on the genitalia, but lies
completely within the sixth abdominal segment. If mgml0 does develop from genital
disc myoblasts, this represents a migration by these myoblasts over a considerable
distance. This muscle could develop from the presumably spatially more immediate
myoblasts within the A6 segment, perhaps the dorsal and/or ventral segmental
myoblasts. Other muscles, like mgm6, mgm8, fgm5 and fgm7, though attaching to
106
the external genitalia, resemble slightly modified versions of segmental muscles and
also may derive from the segmental pools of myoblasts known to give rise to other
segmental abdominal muscles (Bate et al., 1991). twist expression is known to
overlap expression of muscle-specific proteins (Currie and Bate, 1991). It should be
possible, therefore, to follow the development of individual muscles from twist-
expressing cells in the disc through to adult muscles. Such a developmental timecourse would address both the issue of a single or dual primordia and the source of
individual muscles.
It is worth noting that no twist-expressing cells were found associated with the anal
primordium. Muscles associated with the anal plates must be either derived from
myoblasts in the populations of myoblasts associated with the male of female
primordia or from myoblasts invading from outside the disc.
pattern of twist-expressing cells in dsx-dominant mutants
Dominant mutations of the dsx gene result in expression of substantial elements of
both male and female genitalia. In the last chapter we showed that only a subset of
the possible male and female genital muscles developed in association with the male
and female genitalia of dsx' intersexes, a subset containing a total number of around
ten muscles, similar to the number found in each sex. I show here that in dsx'
mutants twist-expressing cells are associated with both the male and female genital
primordia, as in wildtype genital discs (Figures 3.1A and 3.1B and 3.1C).
107
In the last chapter, I proposed that the number of the genital muscles expressed in
dsx mutant intersexes was limited, not by a lack of appropriate insertion sites, but by
a finite number of available myoblasts. In this argument, all available myoblasts are
required for the development of one complete set of genital muscles. In these
intersexual flies, which possess both male and female genitalia, the single pool of
myoblasts would be divided amongst the possible male and female muscles by some
unknown mechanism. If our proposal is correct then XX; dsx'l + larvae would be
expected to a have a similar number of sex-specific myoblasts as wildtype larvae.
More specifically, the number and distribution of twist-expressing cells that give rise
to the mgms and fgms would be expected to be similar between dsx mutant and
wildtype discs. Indeed, the distribution and relative twist-staining properties of
myoblasts aligned with the male and female primordia in the XX; dri'V+ genital
discs were similar to that in wildtype XY and XX genital discs. Without quantitative
analysis, it is not possible to determine whether equal numbers of myoblasts are
distributed in the two populations in mutant and wildtype genital discs, but casual
observation finds obvious differences. However, similarity, or differences of the
particular group(s) of myoblasts destined to form the external genital muscles might
easily go unnoticed against a background of myoblasts destined for the internal
genital, these muscles do not appear to be drastically reduced in the du intersexes.
Irrespective of the complications in separating fgm and mgm precursor myoblasts
from the other twist-expressing myoblasts, no counts of twist-expressing cells have
been done.
108
Future experiments which would address these quantitative questions and follow
the development of the genital muscles, from twist-expressing myoblasts, will clarify
these points.
109
Chapter 4.
A Summary of Results and Conclusions
Thomas J. S. Merritt
110
This project had two main goals: one, to investigate the role, if any, of the dsx
gene in the development of the genital musculature of Drosophila melanogaster, and
two, to identify possible developmental precursors of these genital muscles.
In Drosophila, development of individual sex-specific characteristics is under the
regulation of either of two genetic pathways (for recent reviews see Baker and Ridge,
1980; Baker and Belote, 1983; Baker, 1989; Slee and Bownes, 1990; Steinmann-
Zwicky et al., 1990; Belote, 1992; Burtis and Wolfner, 1992; Cline, 1993). One of
these pathways is dsx-dependent; the dsx gene functions as the output gene of the
pathway. The other pathway is independent of the dsx gene; its output gene is
unknown. The development of the cuticular structures of the genitalia is under the
control of the first pathway (Baker and Ridge, 1980). The development of the only
previously studied sex-specific muscle, the male-specific MOL, is under control of the
second pathway (Taylor, 1992).
I found that the development of the sex-specific
muscles associated with the genitalia and terminal segments is, similar to the cuticular
structures, dependent on dsx activity.
In Drosophila, adult muscles develop de novo from precursors set aside in the
embryo; these precursor cells can be identified by their expression of the twist protein
throughout the larval stage (Bate et al., 1991; Currie and Bate, 1991). I have
identified twist-expressing cells associated with the male and female primordia of the
genital discs of third instar larvae, the imaginal tissues that give rise to the adult male
111
and female genitalia, respectively. These two groups of twist-expressing cells are
qualitatively similar in male, female and intersexual d&x
discs.
Based on these two main findings, and data generated in forming them, I have
drawn several other conclusions, observations and speculations. Here, briefly, is a
summary of the main results and conclusions of the preceding chapters.
1) Adult male and female Drosophila melanogaster each possess a set of 10 muscles
attached to the terminal segments and external and internal genitalia. The set of
muscles found in males was morphologically distinct from the set in females, though
at least some of the muscles appear to be homologous.
2) In flies possessing a reporter gene construct in which the lacZ gene is driven by
the Drosophila 79B actin gene promotor (P[79B- lacZJ), a subset of those muscles
express B-galactosidase by midway through adult development. In males, six of the
genital muscles stained for B-galactosidase. In females, seven of the genital muscles
stained for B-galactosidase. The only other abdominal muscle showing this early
pattern of reporter expression was the Muscle of Lawrence (MOL), the other known
sex-specific muscle.
3) Proper development of the male and female genital muscles is dependent upon
normal dsx activity. Both XX; dsx- and XY; & intersexes developed the same set of
genital muscles. In contrast, MOL development is du-independent; XY; dsx, but not
112
XX; dsx", intersexes develop a MOL.
4) dsx- intersexes develop a subset of the genital muscles seen in wildtype males and
females. This subset contained both distinctly male and distinctly female muscles, but
no intersexual muscles. In total, about one half ( between seven and thirteen) of the
total possible male and female genital muscles (twenty) develop in the intersexes.
This limited number of expressed genital muscles may result from development from
a limiting pool of muscle precursor cells.
5) Expression of the P[79B-lacZ] reporter gene is a separate sex-specific phenotype
from muscle expression. Genital muscles that stained for 11-galactosidase in wildtype
flies did not always stain in either dsx- or dsx'l + intersexes. Staining of most
muscles ranged between less than 50 and 100%; one muscle, fgm10, never stained.
This general reduction in staining frequency may reflect a failure of these muscles to
fully differentiate into their sex-specific phenotype.
6) Muscle fibers spanning between the A7 tergite and sternite sometimes stained for
activity of the P[79B -lacZ] reporter gene construct. The staining may be a product of
transformation of the muscle fibers into a male-like (mgm10) phenotype. This ectopic
muscle staining is much more frequent in dsxD°°31+ intersexes than in dsx- intersexes.
113
The higher frequency of transformation (staining) may represent the activation of a
male phenotype by the ectopic presence of the DSXM protein.
7) The product of the dsx gene acts as a negative regulator in the development of the
genital muscles, more similar to its role in the cuticle (Baker and Ridge, 1980) than
its role in the MOL (Taylor, 1992). The same set of genital muscles found in the ,
loss-of-function mutants, the dsx- intersexes, is found in gain-of-function dsx mutants,
the dst"°/+ intersexes.
8) Two groups of putative myoblasts, identified by their expression of the twist
protein, are present in the genital discs of male and female wandering third instar
larvae. One group is located in the male primordia and the other in the female
primordia. These are likely candidates for the developmental precursors of the genital
muscles. No twist expressing cells are located in the region of the anal primordia.
These two groups of cells are qualitatively similar in discs from XX, XY and XX;
dse"/+ animals.
9) The physical division of the two groups of putative myoblasts is no longer
apparent by the white-prepupal developmental stage. This, and the similarity of the
groups across genotypes suggests that the myoblasts act as a single primordia for both
the male and female muscles.
114
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Appendix
123
Appendix.
Creating fly lines of interest
Construction of a P[7913-lacZ] dsx' recombinant chromosome
Genital muscles of control flies were mapped and identified using differential
interference contrast optics and staining for B-galactosidase in the P[79B actin-lacZ,
?y+] (P[79B- lacZJ) line. To facilitate identification of similar muscles in dsx
intersexes, I needed to construct du' mutants that also had the P[79B-lacZ1 insert. I
approached this problem in two parts; location of the insert and construction of a
P[79B-lacZ1 du- chromosome.
Because dsx- alleles are recessive mutations both chromosomes need to have
mutant alleles for a mutant phenotype to be expressed. If the insert was on the
second chromosome then it would be possible to generate P[79B-lacZI;d,a i Idsx' or
P[79B-1acZI;dsx1IDf(3R)dsx15 flies through simple crosses. If, however, the insert
was on the third chromosome we would have to generate a P[79B-laaj,dsi
chromosome through meiotic recombination. The initial published account
(Courchesne-Smith and Tobin, 1989) placed the P[79B -lacZJ insert in the 72-3 line at
70C, on the left arm of the third chromosome, which on the meiotic maps is at 3-41.
I used simple crosses to definitively locate the insert in the line that I used for the
muscle investigations. I crossed P[79B -laal homozygotes to a double balancer line,
and crossed the F1 heterozygotes to a test line, scoring the F2 generation for the
P[79B -lacZJ insert and balancer chromosomes. Balancers, chromosomes with
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multiple inversions, prevent chromosomes in which recombination has occurred from
being present in oocytes, and were used to prevent recombination between
chromosomes carrying different markers. Flies heterozygous for balancer
chromosomes can be identified by the presence of dominant, visible markers. In this
genetic test, consistent segregation of the visible marker within the p-element
construct from a test balancer chromosome would indicate that the P element was on
the chromosomal homologue of the test balancer; lack of consistent segregation would
indicate that the insert and marker were not on the same chromosome.
A test for the presence of the P-element construct on the second and/or third
chromosome
In the parental cross (figure A.1), P[79B- lacZJ /P[79B -lacZJ flies were crossed to
73/2,7y/MKRS flies. TM2 and MKRS are both third chromosome balancers. TM2
carries a dominant Ultrabithorax allele (Ubx13°); a haltere marker. MKRS carries
Stubble (Sb); a dominant bristle marker. Additionally, both of these balancers were
mutant for the rosy (ry), which effects eye color; the P-element carries a ry+ gene as
a visible marker.
If the insert was on the third chromosome then an F2 fly would never contain both
P[79B-lac2] and the balancer. If the insert was on the second chromosome then both
the balancer and the insert would be present in approximately 25% of the flies. The
insert was followed in two ways. Only flies carrying the insert would have muscles
expressing B-galactosidase, which can be detected by incubation in the X-gal reagent.
Additionally, a more immediate, less invasive, method was possible; the P[79B -lacZJ
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insert carries a ry+ allele, so that only flies with the P[79B-lacZI insert ty+. As well
as being faster than scoring by staining for B-galactosidase, scoring for the insert by
the 07+ phenotype does not require killing the flies, while not a concern in these
crosses, but in later crosses becomes important.
Parental cross: P179B-lacZJIPP9B-lacZ1 (x) an, Ulal" iyIMKRS Sb ry
[select P[79B-lacZJIMICRS]
F1 cross: P[79B-lar.211MKRS (x) rys°6lry"
[score F2 flies for P[79B -lacZJ and Sb]
Figure A.1
Of 39 F2 flies from the F1 cross, 20 were P179B-lacZ 1, ry+ and 19 were Sb. No
flies were found that were both P[79B-lacZ1(ty+) and Sb. The insert segregated from
the balancer; it is on the third chromosome.
To double-check this result I repeated the experiment, crossing P[79B-
lacZJIPP9B-laal flies to In(2LR)Gla /CyO flies (Figure A.2). In(2LR)Gla is an
inversion, marked with the Glazed (Gla) gene, an eye morphology marker, that
suppresses crossing over on the second chromosome. CyO is a second chromosome
balancer marked with the Curly (CY) gene, a wing morphology marker. Because all
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third chromosomes in these flies were ry+, presence of the P[79B -lacZJ insert could
only be detected by staining for B-galactosidase.
Parental cross: P[79B-lacZJIP[79B-laal (x) In(2LR)GlalCy0
[select P[79B-lacZYIn(2LR)Gla]
F1 cross: P[79B-lacZ]/ln(2LR)Gla (x) Canton-S
[score F2 flies for P[79B -lacZJ and Gla]
Figure A.2
Of the 41 flies scored for P[79B-lacZI and Gla
,
9 were Gla alone, 7 were P[79B-
lacZ] alone, 13 were neither Gla nor P[79B -lacZJ and 12 were both Gla and FMBlacZ1. The insert did not segregate consistently from the second chromosome
markers. The insert is not on the second chromosome, thus I had to generate a
recombinant P[79B-lacZ], dsx1 chromosome.
Recombinant crosses to construct the P179B-lacZ1, dsx1 chromosome
To do this I needed to be able to score for the presence of the P[79B -lacZ] insert
without having to kill and stain the fly. The one way to do this is to follow the insert
using the 737+ marker gene, as I did in the crosses to TM2IMKRS, which necessitates a
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ty background. No du-, ry chromosomes were available from the stock centers. I
began by creating a ry third chromosome, with markers to allow us to follow
recombinations in the region of the dsx gene, through meiotic recombination
(Figure A.3).
Parental cross: tys0i611y5'96 (x) ru h st pP cu sr el ru h st p" cu sr e'
[select virgin females]
F 1 cross: 30C:; ru h st pP cu sr elrys°6 (x) ru h st
cu sr el ru h st pP cu sr e'
[select for the recombinant sr,e']
F2 cross: sr elsr e (x) TM2IMKRS
[select ry line and maintain; these flies are rye sr elan]
Figure A.3
ry"6 homozygotes were crossed to a line containing multiple recessive markers to
create heterozygotes (Figure A.3). The females are the flies that were used to
produce the recombinants; that is, gametes in which the rys06 and ru h st pP cu sr e'
chromosomes that had paired, crossed over and recombined to create a
ty",sr,e
chromosome. Females were selected because recombination does not occur in male
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Drosophila. The female heterozygotes were back crossed to ru h st if cu sr elm h st
pP cu sr e males to score for the loss of the markers proximal to the ry gene,
indicating recombination (Figure A.3). Not all of the recombinants were ry", since
sr and ry are 10 map units apart. To find the tys96 recombinants all male sr e' flies
were crossed to TM2IMKRS virgin females (Figure A.3). sr e' males were selected
since they do not have meiotic recombination; preventing recombination of the ry" sr
e' and ru h st pP cu sr e chromosomes.
My next step was to recombine dsx' onto the 75°6 sr e chromosome (Figure A.4).
Both XX and XY dal homozygotes are infertile so I used the closely linked gene pP
to follow dal (da' and pP are separated by only .1 map units), while maintaining a
fertile line.
rys06,sr,e/TM2 males were crossed to pP dsx' females to generate heterozygotes, pP
&xi liy"6 sr e', that were to form the recombinants (Figure A.4). Recombinants were
recovered as pP sr e flies, effectively all of which were pP dal sr e. I used the ru h
st pP cu sr e chromosome, which hasp?, to follow the &x' gene. This does not,
however, allow me to score for ty. Individual recombination events could have been
either proximal or distal to the ry locus, only proximal recombinations would give me
a pP dsx' ry30'6 sr e' chromosome. To test for the presence of ry"6, all pP sr e' /ru h st
pP cu sr e' males were crossed to the 7312IMKRS line (Figure A.4). Again, males
were used to prevent further recombination of the pP dsx' sr e chromosome.
Recombination of the P[79B -lacZJ insert onto this dsx' chromosome could be
followed using the ly+ phenotype. Finally, I crossed dsx' rys°6 flies to P[79B -lacZJ
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homozygotes and then crossed the resulting female heterozygotes, XX;pP dsx1 rys" f
elP[79B-lacZI, to the rys" sr e line to score for ry+ recombinants (Figure A.5).
The rys016 sr e chromosome allowed me to score for recombinants that had picked
up the insert; only these flies were ry+ sr e. Using this chromosome I was not able
to score for f d&, but only recombinants proximal to pP would give me the P[79BlacZ], dsx' chromosome. To score for pP, and thereby d&, all male ry+ sr e flies
were individually crossed to virgin female pP ds.11 ty306 sr eITM2. All lines that
produced dsx- intersexes had both the P[79B -lacZJ insert and the dsxi gene.
Parental cross: 7375°6 sr eITM2 (x) pP ds.r1ITM6b
[select Ubx13D +, TY' virgin females]
F1 cross: XX;pP da 110,5°6 sr e (x) ru h st pP cu sr e' /ru h st pP cu sr e [select
pP,sr,e]
F2 cross: XY;pP dsxl sr elru h st pP cu sr e (x) TM2IMKRS
[select ry: pP dsxl ry"6 sr el7M2]
Figure A.4
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Two lines were found to have the 13[79B-lacZ] if drx' 73,566 sr el chromosome.
These were maintained over the balancer TM2.
Parental cross: pP dsx" rys°6 sr elan (x) P[79B-lacZJIPP9B-laal
[select virgin female Ubx13"]
pP dal ty5°6 sr elP179B-lacZ1 (x) ry5°6 sr el7312
[select ty+ sr e']
XY;P[79B -lacZJ 73,506 sr eh)," sr e' (x) pP dui 737506 sr e'/TM2
[select dsx' ty+]
Figure A.5
Creating a y/y+Y:P17913-lacZ1 dsx' line:
I now had the P[79B -lacZJ dsx' line I set out to create. Homozygotes were
intersexual with staining genital muscles. In addition to comparing muscles between
controls, dominant dsx' mutants and recessive dsx mutants I was also interested in
comparing XX and XY recessive dsx mutants. I could distinguish XX and XY du
intersexes in three ways. These intersexes are phenotypically very similar, but can be
distinguished by expression of a fifth segment male-specific muscle, the Muscle of
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Lawrence (MOL). MOL expression is independent of du control;XY, but not XX,
du mutants express a MOL. In addition I used a chromosomal indicator of sex.
Parental cross: y/y+Y;pP dsxl/TM6 (x) X/Y;TM2 /MKRS
[select ylX,pP dsx'/TM2 virgins and Xly+Y;MKRSIpP dui
F1 cross: ylX;pP dsx'/TM2 (x) yly+Y;pP dalITM6B
[select yly;pP dsx' /TM2 virgin females]
F2 cross: ylye du 1 17M2 (x) Xly+Y;MKRSIpP dui (from the first cross)
[select yly+Y;73121MKRS]
F3 cross: yly+Y;7312IMKRS (x) X/y;TM2 /MKRS
[select yly;TM2IMKRS]
F4 cross: yly;7312IMKRS (x) yly+Y;7312IMKRS (from the third cross)
[maintain as a line]
Figure A.6
Two different tactics were used. In creating the dsx'llDf(3R)dsx1.5
transheterozygote I crossed XY;P[79B -lacZJ dsx' /TM2 flies to yly,Df(3R)dul5ITM6B
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flies. y,yellow, is a cuticle pigment marker. All Fl XY flies were y/Y and therefore
yellow; XX flies were y/X and had wild type pigment.
I determined chromosomal sex in P[79B-laaJdxri1 homozygotes by creating a
y/y+Y line of flies through standard crosses. A y+Y is carries the wild type yellow
gene, normally found on the X chromosome. y/y+Y males have wild type
pigmentation.
I first created a y/y+Y; TM2 /MKRS line from a yly+Y;i1 dsa-lITM6 line and the
TM2 /MKRS line (Figure A.6). In these crosses the pP dsi/TM6 chromosomes were
not important, this line I used only as a source of the y and y+Y chromosome.
Parental cross: 3C/X; P[79B-lacZ1 pP dsx' ty"6 sr eITM2 (x) y/y+Y;TM2IMKRS
[select virgin female TM2 flies]
Fl cross: ylX;P[79B-lacZ] pP dsx' ry" sr elTM2 (x) yly+Y;73121MKRS [select y
TM2 virgin females]
F2 cross: yly;P[79B-lacZ1 pP dsx' tys06 sr exalt/12 (x) y/y+Y;TM2IMKRS
[select male and female TM2 and maintain as a line]
Figure A.7
Finally I generated a stable, yly+Y;P[79B-lacZ] dsx' line through a series of
crosses between the y/y+Y;TAI2IMKRS line and the P[79B -lacZJ dsx' lines (Figure
133
A.7).
This was the last line I generated; a yly+Y;P[79B-laal dal line in which the
homozygotes develop as intersexes, distinguishable as XX or XY, with P179B-lacZ]
staining. The two P[79B -lacZJ dal lines were maintained both as unmarked XY and
y/y+Y lines.
Crosses involving dominant dsx alleles
I also wished to use the P[79B -lacZJ insert to aid our identification of genital
muscles in flies expressing dominant alleles of dsx. Because the dominant du alleles
are expressed in heterozygotes and a single copy of the P[79B-lacZ1 insert is
sufficient for robust staining, a single cross between dap males and females
homozygous for the P[79B -lacZJ insert gave us du mutants with muscle staining
(Figure A.8).
The Y chromosome in these crosses carries the Bar stone (Be) allele, an adult eye
morphology marker, only XX flies were B` +. Additionally, in the line we used dse
is maintained over TM6B, a balancer chromosome with a dominant marker Tb
(Tubby), a body shape marker detectable in all stages of the Drosophila life cycle.
XX;P[79B-lacZ] de intersexes were Br+ Tb+ and could, therefore, be distinguished
from de flies as larvae, pupae and adults.
I also looked at the muscles in flies mutant for another dominant dsx allele, dsxM
mutants, using a similar cross.
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Parental cross: ITY;dte Sb e'ITM6B,Tb (x) XIX;P[79B-laMIPP9B-lacZ]
[select B'+ Tel
Parental cross: 13s Y ;dam I TAI6B ,Tb (x) X/X;P[79B- lacZJIP[79B -lacZJ
[select /3"- Tbl
Figure A.8
XX flies heterozygous for a dominant dsx allele and a recessive dsx allele, or a
dsx- deficiency, develop as somatic males. To check that the genital muscles in these
pseudomales developed and stained as in wildtype males we crossed de or dsx'
males to PP9B-lacZ1 pp dal ry°6 sr eITM2 females (Figure A.9). Pseudomales were
identified as Rs+, Ubxu° +,Tb+. Additionally, I checked muscle development, but not
staining, in ds.e/Df(3R)dsx15 pseudomales, generated through a similar
cross (Figure A.9)
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Parental cross: BIY;dse Sb el7M6B,Tb (x) XJX;P[79B-laal pP dsx' rys06 sr eITM2
[select Sb Bs+ Ubx13" Tb+]
Parental cross: BsY;delTM6B,Tb (x) XIX;P[79B-laa] if dsx' lys06 sr eITM2
[select /3f+ Ubx13" Tb+]
Parental cross: FY;da-D Sb el7M6B,Tb (x) yly,Df(3R)dal517M6B
[select Sb Bs' Tb+]
Figure A.9